Treating stress response with chemokine receptor ccr5 modulators

ABSTRACT

The use of chemokine receptor CCR5 modulators (e.g., CCR5 antagonists) is disclosed for the treatment or prevention of stress response (e.g., fever) in a subject resulting from surgery, infection or other insult to the subject (e.g., a warm-blooded vertebrate). Methods for treating or preventing disorders involving the activation of pro-inflammatory cytokines by administration of a CCR5 modulator, for inhibiting the endogenous production of pro-inflammatory cytokines by the administration of CCR5 modulators, and for determining the efficacy of CCR5 modulators in correcting abnormal levels of pro-inflammatory cytokines are also disclosed.

This application claims the benefit of U.S. Provisional Application No.60/350,868, filed Jan. 22, 2002, and U.S. Provisional Application No.60/365,097, filed Mar. 18, 2002, the disclosures of which are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to therapeutic methods foralleviating stress responses. More particularly, the present inventionrelates to the administration of a chemokine receptor CCR5 modulator(e.g., a CCR5 receptor antagonist) to treat a subject suffering from astress response (e.g., fever and malaise as a result of infection and/orinjury) or to prevent a stress response in a subject at risk therefor (apre-operative surgical patient). The present invention also relates tothe administration of CCR5 modulators for the treatment or prevention ofthe inflammatory response mediated by pro-inflammatory cytokines.

BACKGROUND OF THE INVENTION

Physical injury, wounds, surgery, burns, infection, and other insults tothe body typically result in a stress response that can include fever,pain, headache, inflammation, malaise, and/or other conditions. Thestress response can sometimes be beneficial in that it can be part ofthe body's immune response to the insult and a tool in the restorationof health. For example, fever in response to a bacterial infectiontypically will retard the growth of the bacterial invader and at thesame time increase the bactericidal activity of neutrophils andmacrophages responding to the infection (Netea et al., ClinicalInfectious Diseases 2000, 31: S178-S184). Unfortunately, the body'sresponse to an insult is in many circumstances harmful, leading todangerously high fever, severe inflammation, tissue destruction, etc.,which can impede recovery and even lead to shock or death. The fever andmalaise that typically follow surgery, for example, can causesignificant post-operative discomfort without contributing to recovery.

Physical injury, surgery, infection, and other traumatic insults, aswell as a variety of other immunological disorders, provoke abiochemical cascade of pathophysiologic events that is triggered byexcessive tissue or blood concentrations of pro-inflammatory cytokinesreleased or synthesized following the insult. The pro-inflammatorycytokine, interleukin-1 (IL1), is at the head of many inflammatorycascades and its actions, often via the induction of other cytokines,lead to activation and recruitment of leukocytes into damaged tissue,local production of vasoactive agents, and hepatic acute phase response.That it is a key mediator in the inflammatory response is evident fromthe description set forth in Dinarello, Blood 1991, 77: 1627-1652;Dinarello et al., New England J. Med. 1993, 328: 106-113; and DinarelloFASEB J. 1994, 8:1314-1325.

IL1 is produced by a number of cell types, including monocytes andmacrophages. When locally released, IL1 diffuses into the circulation,where it is ultimately carried to the hypothalamus. There, it acts tostimulate the production of prostaglandin-E which acts as aninflammatory mediator. IL1 has also been shown to be an endogenous humanpyrogen, which produces fever (Ikejima et al., J. Clin. Invest. 1984,73: 1312). Two forms of IL1 have been isolated, IL1-α and IL1-β. IL1 isknown to incite a variety of other systemic responses; e.g., itmobilizes neutrophils, stimulates liver production of acute phaseproteins and complements, and is also responsible for the increases incirculating eicosanoid levels, levels of interleukin-6 and levels oftumor necrosis factor (Dinarello, Rev. Infect. Disease, 6: 51-94).

Interleukin-6 (IL6) is a multi-functional cytokine that plays a pivotalrole in host defense mechanisms (Heinrich et al., Biochem. J. 1990, 265:621; Van Snick, J. Annu. Rev. Immunol. 1990, 8: 253; and Hirano et al.,Immunol. Today 1990, 11: 443). However, examples of disorderscharacterized by elevated serum levels of IL6 in patients abound, andoverproduction of IL6 has been implicated in sequellae oftransplantations, autoimmune diseases and, in particular, certain typesof septicemia. Indeed, the overproduction of IL6 has been suggested toplay a role in the pathogenesis of the above referenced diseases (Hiranoet al., Immunol. Today 1990, 11: 443; Sehgal, Proc. Soc. Exp. Biol. Med.1990, 195: 183; Grau, Eur. Cytokine Net 1990, 1: 203; Bauer et al., Ann.Hematol. 1991, 62: 203; Campbell et al., J. Clin. Invest. 1991, 7: 739;and Roodman et al., J. Clin. Invest. 1992, 89: 46).

IL6 induction rapidly follows injury or other insult. Plasma levels ofIL6 can be detected as early as 30 minutes after incision in patientsundergoing elective surgery (Shenkin et al., Lymphok. Res. 1989, 8:123-127). Maximal levels of IL6 are found between 90 minutes and 6 hourspost surgery (Pullicino et al., Lymphok. Res. 1990, 9: 2-6; Shenkin,Lymphok. Res. 1989, 8: 123-127). In contrast, upon exposure to aninfectious agent, elevated plasma levels may persist for days (Bauer, J.et al., Ann. Hematol. 1991, 62: 203-210). Importantly, elevated serumlevels of IL6 have been observed in transplant rejection andinflammatory bowel disease (van Oers et al., Clin. Exper. Immunol. 1988,71: 314-319; Bauer et al., Ann. Hematol. 1991, 62: 203-210).

While numerous approaches to regulate the production of interleukin-6have been proposed, no substance or method has been reported to inhibitspecifically the production of IL6 or to effectively block its adverseactions. In fact, no natural receptor-antagonist for IL6 has so far beenidentified.

The tumor necrosis factor (TNF) family is an expanding set ofextracellular signaling molecules (ligands) with biological activitiesthat are intimately associated with a variety of disease conditions.There are several disease states in which excessive or unregulated TNFproduction by monocytes, macrophages or related cells are implicated inexacerbating and/or causing the disease. For example, TNF, theprototypic member of this family, is well known as a mediator of septicshock, inflammation, and graft-versus-host disease. (See, for example,Cerami, Immunol Today 1988, 9: 28-31; Revel, Ciba Found. Symp. 1987,129: 223-233; Cohen, J. Bone Marrow Transplant. 1988, 3(3): 193-197).

Septic shock is a life-threatening complication of bacterial infections.It results from the uncontrolled, sequential release of mediators havingpro-inflammatory activity following infection with gram-negativebacteria, and in response to endotoxins (see, e.g., Tracey et al,Science 1986, 234: 470; Alexander et al, J. Exp. Med. 1991, 173: 1029;Doherty et al, J. Immunol. 1992, 149: 1666; Wysocka et al., Eur. J.Immunol. 1995, 25: 672). Endotoxin exerts its effect by inducing potentmacrophage activation, and release of cytokines such as TNF-α, IL1, IL6,IL12, and interferon-gamma (IFN-γ) (see Van Deuren et al, J. Pathol.1992, 168: 349). Two key mediators of septic shock are TNF and IL1,which are released by macrophages and appear to act synergistically tocause a cascade of physiological changes leading to circulation collapseand organ failure (Bone, Ann. Intern. Med. 1991, 115: 457-469).Overproduction of IL6 has also been linked to septic shock (Starnes,Jr., et al., J. Immunol. 1990, 145: 4185). The central role ofpro-inflammatory cytokines in the pathogenesis of septic shock (alsoreferred to as endotoxic shock) is underlined by the occurrence of highlevels of circulating cytokines in both humans and experimental animalsduring endotoxemia (see Stevens et al., Curr. Opin. Infect. Dis. 1993,6: 374). Significantly, a substantial body of literature shows thatanti-cytokine action can improve the outcome of subjects challenged byendotoxin or gram-negative bacteria (see Beutler et al., Science 1985,229: 689, and Heinzel et al., J. Immunol. 1990, 145: 2920). For example,Bozza et al., J. Exp. Med. 1999, 189: 341 teaches targeting of genesencoding pro-inflammatory cytokines, and Ohlsson et al., Nature 1990,348: 550 teaches administration of IL1 receptor antagonists.

Pro-inflammatory cytokines including IL1, IL6 and TNF mediate thecondition known as sepsis in substantially the same manner as septicshock. Sepsis—also known as septicemia, septic syndrome and septicresponse—has no standard definition, but typically refers to severesystemic infection. The traditional agents for sepsis were gram-negativebacteria, but more recently patients have been observed withcharacteristic responses of sepsis without a clearly identifiableinciting microbe. The term sepsis has thus come to be associated withany systemic response to overwhelming infection or other severe insult(Kelly et al, Ann. Surg. 1997, 225(5): 530-541, see esp. 542-543).Despite the major advances of the past several decades in the treatmentof serious infections, sepsis remains a serious health threat (S. M.Wolff, New Eng. J. Med. 1991, 324: 486-488).

The foregoing observations have buttressed the importance of regulatingpro-inflammatory cytokine production for the maintenance of thehomeostasis of immune system in a human body and for the treatment andprophylaxis of pathologies attending a post surgical inflammatoryresponse. Thus there remains a need for an effective, clinicallyapplicable approach for preventing or treating a post-surgical stressresponse/inflammatory response and cytotoxic T-lymphocyte (CTL) and/orcomplement-dependent rejection of organ or tissue transplants.

Stress response accompanying physical injury, surgery (including tissueand organ transplantation), infection and other insults has been treatedwith some success with various drugs. Corticosteroids andimmunosuppressants are often employed, for example, but these agentshave serious, well known side effects. Non-steroidal antiinflammatorydrugs (NSAIDs) are also employed, but they are known to cause gastriculceration. There is a need for new drugs for the therapeutic orprophylactic treatment of stress response. More particularly, there is aneed for agents that will reduce or suppress the activation ofinflammatory cells or their production of pro-inflammatory cytokines inresponse infection (especially serious and/or systemic infection), majorsurgery, allograft transplant rejection, physical injury, and othertraumatic insults.

The following references are of interest as background:

WO 00/76972 discloses N-cyclopentyl modulators of the activity ofchemokine receptors including CCR5.

WO 01/78707 discloses a method of treating the rejection of transplantedgrafts by administration of an antagonist of CCR5 function to the graftrecipient.

Leon, J. Appl. Physiol. 2002, 92: 2648-2655 is a review of the cytokineregulation of fever that discusses the role of IL1, IL6, TNF-α, and IL10in the inducement and inhibition of fever.

SUMMARY OF THE INVENTION

The present invention is directed to the use of chemokine receptor CCR5modulators (e.g., CCR5 antagonists) to treat or prevent stress response(e.g., fever) in a subject resulting from a planned (e.g., surgery) orunforeseen (e.g., injury due to an accident) insult to the subject. Thepresent invention also includes a method for treating or preventing adisorder involving the activation of pro-inflammatory cytokines byadministration of a CCR5 modulator. The present invention furtherincludes a method for inhibiting the endogenous production ofpro-inflammatory cytokines by the administration of CCR5 modulators. Thepresent invention still further includes a method for determining theefficacy of CCR5 modulators in correcting abnormal levels ofpro-inflammatory cytokines.

Other embodiments, aspects and features of the present invention areeither further described in or will be apparent from the ensuingdescription, examples and appended claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes methods for treating or preventing astress response via administration of a therapeutically effective amountof a chemokine receptor CCR5 modulator to a subject suffering from astress response or at risk to suffer from a stress response. Moreparticularly, the present invention includes a method of treating orpreventing stress response in a subject in need thereof, which comprisesadministering a therapeutically effective amount of a CCR5 antagonist tothe subject. Treatment or prevention of the stress response can hastenthe subject's return to normal activity with a reduced requirement fornarcotic analgesics and/or with a lower complication rate. In anembodiment of this method, the subject is a warm-blooded vertebrate. Inan aspect of this embodiment, the subject is a primate, and isespecially a human. In another embodiment of the method, the subject isa surgical patient who has pre-existing infection (e.g., sepsis fromabscess or empyema) or inflammation (e.g, rheumatoid arthritis or acutemyocardial infarction). In still another embodiment of the method, thesubject is a cardiac surgery patient. In an aspect of this embodiment,the cardiac surgery patient is a patient who has recently experienced amyocardial infarction or who has a lung infection or liver disease.

Another embodiment of this method is a method of treating or preventingstress response in a subject in need thereof, which comprisesadministering a therapeutically effective amount of a CCR5 antagonist tothe subject, wherein the stress response comprises inflammation andassociated pain and/or malaise resulting or expected from a plannedstress. In an aspect of this embodiment, the planned stress is surgery.The surgery can be any surgical procedure including, but not limited to,major surgery such as cardiac surgery, a minor outpatient procedure, andminimal access surgery (also known in the art as minimally invasivesurgery) such as laparoscopy or thoracoscopy. The method can reduce orpreclude the delay in the return of normal respiratory and bowelfunction that is often observed in surgical patients.

As used herein, the term “stress response” refers to any response (i.e.,physiological change) seen in a subject exposed to an insult (which mayalternatively be referred to as a stressor). An insult is a trauma(e.g., physical injury, wounds, surgery, burns) or a physiopathologicalstate (e.g., infection such as bacteremia, endotoxin infusion) thatresults in changes to existing rhythmical processes which arehomeostatic in nature. Stress response includes, but is not limited to,any one or more of the following conditions: hyperthermia, hypothermia,hypertension, hypotension, inflammation, malaise (i.e., discomfort ordebility typically characterized by decreased activity and/or loss ofappetite), shock (e.g., septic shock), tissue damage, organ damageand/or failure, and sepsis. Fever and malaise, for example, aretypically seen in mammals after organ transplantation using conventionalimmunosuppression.

The term “treating”, or a variant thereof (e.g., “treatment”), refers toreducing or ameliorating an existing undesirable or adverse condition,symptom or disease (e.g., stress response due to exposure to a stressor)or delaying its onset in a subject in need of such reduction,amelioration or delay.

The term “preventing”, or a variant thereof (e.g., “prevention”), refersto prophylaxis of an undesirable or adverse condition, symptom ordisease in a subject who is at increased risk of acquiring such acondition, symptom, or disease as a result of being subjected or exposedto an insult. “Increased risk” means a statistically higher frequency ofoccurrence of the condition, symptom, or disease in the subject as aresult of the insult in comparison to the frequency of its occurrence inthe general population (e.g., an individual about to have surgery wouldbe at a substantially increased risk for hyperthermia and inflammationsubsequent to the surgery).

The term “subject” as used herein refers to any vertebrate species whichis the object of treatment, observation or experiment with respect tothe present invention. In one embodiment, the subject is a warm-bloodedvertebrate, particularly a mammal, preferably a primate, and morepreferably a human. A mammal is understood to include any mammalianspecies in which treatment or prevention is necessary or desirable,particularly agricultural and domestic mammalian species. Thus, subjectscontemplated for the present invention include primates (includinghumans), as well as those mammals of importance due to being endangered(such as Siberian tigers), of economic importance (animals raised onfarms for human consumption) and/or social importance (animals kept aspets or in zoos) to humans, such as cats, dogs, swine (e.g., pigs, hogs,and wild boars), ruminants (e.g., cattle, oxen, sheep, giraffes, deer,goats, bison, and camels), and horses. Birds are also contemplated assubjects in the present invention including birds that are endangered,kept in zoos, as well as fowl, and more particularly domesticated fowl(e.g., poultry, such as turkeys, chickens, ducks, geese, guinea fowl,and the like) of economic importance to humans.

The term “patient” refers to a subject as defined above who/which isawaiting or receiving medical care or is or will be the object of amedical procedure (e.g., surgery).

The term “cardiac surgery patient” refers to a patient who has or willhave open heart surgery using cardiopulmonary bypass. “Cardiopulmonarybypass”, or a variant thereof (e.g., “bypass” or “circulatory bypass”)refers to circulatory bypass of the heart and lungs; i.e., the conditionwherein the heartbeat is stopped for the purpose of surgery on the stillheart, and the blood supply to the brain and the remainder of the body,excluding the heart and lungs, is provided by an extracorporeal machinethat oxygenates and pumps the blood.

The term “transplant” refers to the grafting, implantation ortransplantation of organs, tissues, cells (e.g., bone marrow) and/orbiocompatible materials onto or into the body of an animal. The termencompasses the transfer of tissues from one part of the animal's bodyto another part and the transfer of organs, tissues, and/or cellsobtained from a donor animal (either directly or indirectly such as anorgan or tissue produced in vitro by culturing cells obtained from theanimal) into a recipient animal. The animal is suitably a warm-bloodedvertebrate, is typically a mammal, and is especially a primate (e.g., ahuman). The term “transplant rejection” means any immune reaction in therecipient directed against grafted organs, tissues, cells, and/orbiocompatible materials.

The term “therapeutically effective amount” (or more simply an“effective amount”) as used herein means that amount of active agent oractive ingredient (e.g., chemokine receptor CCR5 modulator, especially aCCR5 antangonist) that elicits the biological or medicinal response in atissue, system, animal or human that is being sought by a researcher,veterinarian, physician or other clinician, which includes alleviationor prophylaxis of the symptoms of the disease or condition being treatedor prevented. When the salt of a chemical compound is administered,references to the amount of active ingredient are to the free acid orfree base form of the compound. Actual dosage levels of activeingredients in a composition employed in a method of the presentinvention can be varied so as to administer an amount of the activecompound(s) that is effective to achieve the desired therapeuticresponse for a particular subject and/or application.

The term “administration”, or a variant thereof (e.g., “administering”),means providing the active agent or active ingredient (e.g., a CCR5antagonist), alone or as part of a pharmaceutically acceptablecomposition, to the subject (e.g., warm-blooded vertebrate) inwhom/which the condition, symptom, or disease is to be treated orprevented.

By “pharmaceutically acceptable” is meant that the ingredients of apharmaceutical composition are compatible with each other and notdeleterious to the recipient thereof.

The present invention also includes a method of treating or preventinghyperthermia in a subject in need thereof, which comprises administeringa therapeutically effective amount of a CCR5 antagonist to the subject.Embodiments of this method include the method as just described whereinthe subject is a warm-blooded vertebrate, or is a primate, or is ahuman. Other embodiments of this method include the method as originallydescribed wherein the subject is other than a graft transplant patient,or is a cardiac surgery patient.

The present invention also includes a method of treating or preventinghypothermia in a subject in need thereof, which comprises administeringa therapeutically effective amount of a CCR5 antagonist to the subject.Embodiments of this method include the method as just described whereinthe subject is a warm-blooded vertebrate, or is a primate, or is ahuman. Other embodiments of this method include the method as originallydescribed wherein the subject is other than a graft transplant patient,or is a cardiac surgery patient.

The term “hyperthermia” refers herein to the elevation of thetemperature of a subject's body, or a part of a subject's body, comparedto the normal temperature of the subject. In mammals, a normal bodytemperature is ordinarily maintained due to the thermoregulatory centerin the anterior hypothalamus, which acts to balance heat production bybody tissues with heat loss. The terms “fever” and “hyperthermia” aresometimes distinguished from each other, wherein fever refers to aregulated elevation in a subject's thermal set point (in response, e.g.,to an infection or other insult), and hyperthermia refers to anunregulated rise in body temperature that is not triggered by anincreased thermal set point but is instead in response to an internal(e.g., exercise) or external (e.g., hot ambient conditions) source ofheat. The terms “fever” and “hyperthermia” are used interchangeablyherein, and both refer to a regulated rise in body temperature inresponse to an insult or other inflammatory stimulus.

The term “hypothermia” refers to a decrease in the temperature of asubject's body, or a part of a subject's body, compared to the normaltemperature of the subject. The decrease is typically a regulateddecrease in the subject's thermal set point, such as in response to aninsult (e.g., infection).

Chemokines are a family of pro-inflammatory mediators that promoterecruitment and activation of multiplelineages of leukocytes (e.g.,lymphocytes, macrophages). They can be released by many kinds of tissuecells after activation. Continuous release of chemokines at sites ofinflammation can mediate the ongoing migration and recruitment ofeffector cells to sites of chronic inflammation. The chemokines arerelated in primary structure and share four conserved cysteines, whichform disulfide bonds. Based upon this conserved cysteine motif, thefamily can be divided into distinct branches, including the C—X—Cchemokines (a-chemokines), and the C—C chemokines (P-chemokines), inwhich the first two conserved cysteines are separated by an interveningresidue, or are adjacent residues, respectively (Baggiolini, M. et al.,Immunology Today 1994, 15: 127-133).

The C—X—C chemokines include a number of potent chemoattractants andactivators of neutrophils, such as interleukin 8 (IL-8), PF4 andneutrophil-activating peptide-2 (NAP-2). The C—C chemokines include, forexample, RANTES (Regulated on Activation, Normal T Expressed andSecreted), macrophage inflammatory proteins 1-alpha and 1-beta (MIP-1αand MIP-1β), eotaxin and human monocyte chemotactic proteins 1 to 3(MCP-1, MCP-2, MCP-3), which have been characterized as chemoattractantsand activators of monocytes or lymphocytes. Chemokines, such as IL-8,RANTES and MIP-1α, for example, have been implicated in human acute andchronic inflammatory diseases including respiratory diseases, such asasthma and allergic disorders.

The chemokine receptors are members of a superfamily of Gprotein-coupled receptors (GPCR) which share structural features thatreflect a common mechanism of action of signal transduction (Gerard, C.et al., Annu Rev. Immunol. 1994, 12: 775-808; Gerard, C. et al., Curr.Opin. Immunol. 1994, 6: 140-145). Conserved features include sevenhydrophobic domains spanning the plasma membrane, which are connected byhydrophilic extracellular and intracellular loops. The majority of theprimary sequence homology occurs in the hydrophobic transmembraneregions with the hydrophilic regions being more diverse. The receptorsfor the C—C chemokines include: CCR1 which can bind, for example,MIP-1α, RANTES, MCP-2, MCP-3, MCP-4, CKbeta8, CKbeta8-1, leukotactin-1,HCC-1 and’ MPIF-1; CCR2 which can bind, for example, MCP-1, MCP-2, MCP-3and MCP-4; CCR3 which can bind, for example, eotaxin, eotaxin-2, RANTES,MCP-2, MCP-3 and MCP-4; CCR4 which can bind, for example, TARC or MDC;CCR5 which can bind, for example, MIP-1α, RANTES, MIP-1β, MCP-1, MCP-2and MCP-4; CCR6 which can bind, for example, LARC/MIP-3α/exodus; CCR7which can bind, for example, ELC/MIP-3β; CCR8 which can bind, forexample, I-309; CCR9 which can bind, for example, TECK; and CCR10 whichcan bind, for example, ESkine and CCL27 (Baggiolini, M., Nature 1998,392: 565-568; Luster, A. D., New England J. Med. 1998, 338(7): 436-445;Tsou et al., J. Exp. Med. 1998, 188: 603-608; Nardelli et al., J.Immunol. 1999, 162(1): 435-444; Youn et al., Blood 1998, 91(9):3118-3126; Youn, et al., J. Immunol. 1997, 159(11): 5201-5205; Zaballoset al., J. Immunol. 1999, 162: 5671-5675; Jannin et al., J. Immunol.2000, 164: 3460-3464; Homey et al., J. Immunol. 2000, 164: 3465-3470).The receptors for the CXC chemokines include: CXCR1 which can bind, forexample, IL-8, GCP-2; CXCR2 which can bind, for example, IL-8,GROalpha/beta/gamma, NAP-2, ENA78, GCP-2; CXCR3 which can bind, forexample, interferon gamma (IFNγ)-inducible protein of 10 kDa (IP-10),monokine induced by IFNγ (Mig), interferon-inducible T cellchemoattractant (I-TAC); CXCR4 which can bind, for example, SDF-1; andCXCR5 which can bind, for example, BCA-1/BLC (Baggiolini M., Nature1998, 392: 565-568; Lu et al., Eur. J. Immunol. 1999, 29: 3804-3812).

An aspect of the present invention is that blockade of hyperthermicresponse to stress (e.g., surgical stress and/or graftischemia/reperfusion injury) and of hypothermic response to stress canbe provided by CCR5 inhibition. The term “inhibition” (or “inhibiting”)refers to the reduction or suppression of a given condition, symptom, ordisease, wherein in this case the condition is due to the activity ofthe CCR5 receptor. While not wishing to be bound by any particulartheory of operation, it is believed that CCR5 and associated chemokines(MIP-1α, MIP-1β and RANTES) mediate the cytokine-driven innate immuneresponse to stress. As described more fully in Example 1 below, studiesof the stress response in monkeys following cardiac allotransplantationhave been performed. In the studies, twelve monkeys received cardiacallografts. Five were treated with a CCR5 antagonist (only) beginning attransplant, two were treated with a CCR5 antagonist combined withcyclosporin beginning at transplant, and three received only salineinfusion (“control”). Two animals were treated with cyclosporin only.Six out of the seven monkeys treated with a CCR5 antagonist did notdevelop a fever (i.e., a temperature greater than 38.5° C.) whilerecovering from the transplantation procedure, and had an averagetemperature over the first three days after surgery (circa 37.5° C.)that was about one degree lower than the control monkeys (ca. 38.5° C.).The average temperature in the CCR5 antagonist-treated animals was about0.5° C. lower than in the cyclosporin-only-treated animals (ca. 38.0°C.). The CCR5 antagonist-treated monkeys did not exhibit malaise andbehaved as if they had not had surgery. The CCR5 antagonist-treatedmonkeys also did not exhibit typical symptoms of abdominal tenderness,which would otherwise have been expected, either initially aftertransplant, or subsequently during rejection of the graft. Absence ofmalaise and graft abdominal tenderness was unexpected, and was observeddespite the occurrence of major biochemical perturbations (i.e.,increased creatinine and bilirubin) that normally have been associatedwith malaise. In one instance these biochemical perturbations resolvedduring ongoing therapy in an animal whose rejected heart was removed,indicating that the biochemical perturbations were not due to the CCR5antagonist but rather were due to transplant rejection. The animals alsorecovered more quickly from surgery as judged by how quickly theyresumed normal activity levels, despite performance of multipleadditional procedures on days 4 and 7-8 after the initial transplant.

Other agents (e.g., steroids, which block NFkB; non-steroidalanti-inflammatory drugs such as acetaminophen, aspirin (NFkB+COXinhibition), COX-inhibitors) block some consequences of inflammationsuch as fever. However, they do not reliably prevent importantsequellae, such as local and systemic capillary leak, cellularinfiltration of tissues, localized pain and systemic malaise, andtransient impairment of organ function. Thus, CCR5 blockade can be auseful adjunctive or alternative therapy to steroidal or non-steroidalanti-inflammatory agents for reducing pain, suffering, and inhibition oforgan function (e.g., lung and bowel function) associated with thestress of surgery (including major surgery and minimal access surgery),trauma other than surgery (e.g., burns or physical injury), or acuteillness, and for control of inflammation in general medical practice.

An aspect of the present invention is that a CCR5 modulator (e.g., aCCR5 antagonist) can ameliorate or block fever (hyperthermia), such asfever resulting from the innate immune response, or hypothermia. Whilenot wishing to be bound by any particular theory of operation, it isbelieved that CCR5 antagonists can reduce or suppress the elaboration ofmediators of inflammation, especially the pro-inflammatory cytokinesIL1, IL6 and TNF and most especially IL1 and IL6, and thereby reduce orblock fever (or hypothermia) and other symptoms associated with theirrelease in response to an insult.

The present invention also includes a method for treating or preventingstress response in a subject in need thereof, which comprisesadministering to the subject a CCR5 antagonist in an amount effective toinhibit endogenous production of one or more pro-inflammatory cytokinesselected from the group consisting of IL1, IL6, and TNF (e.g., one ormore cytokines selected from the group consisting of IL1 and IL6). Anembodiment of this method is the method as just described, except thatthe stress response is stress response to surgery. Another embodiment ofthis method is the method as originally described, except that thestress is hyperthermia, and is especially surgical hyperthermia (i.e.,hyperthermia which arises as a result of surgery). In an aspect of eachof the foregoing embodiments, the subject is other than a grafttransplant patient. Another embodiment of this method is the method asoriginally described, except that the stress is hypothermia, such assurgical hypothermia (i.e., hypothermia which arises as a result ofsurgery).

References herein to IL1, IL6 and TNF are understood to include thevarious isoforms of each of the cytokines; e.g., IL1-α., IL1-β, TNF-α,and TNF-β. Thus, for example, inhibition of the endogenous production ofIL1 is understood to include inhibition of either one or both of itsisoforms.

The term “inhibiting the endogenous production” of one or more of thecytokines IL1, IL6 and TNF means: (a) decreasing excessive in vivolevels of the cytokine in the subject (e.g., human) to normal levels forseveral types of cells, including but not limited to monocytes and/ormacrophages; (b) down regulating in the subject's tissue (e.g., humantissue) an excessive in vitro or in vivo level of the cytokine to normallevel; or (c) down regulating the cytokine to a normal level by reducingor suppressing direct synthesis of the cytokine as a post-translationevent.

The normal level of cytokine can vary from one subject to the next (see,e.g., Roth-Isigkeit A. et al., Clin. Exp. Immunol. 2001, 125: 80-88).Accordingly, in the case of a planned insult such as surgery, it ispreferred to determine the normal cytokine level for the given subjectprior to the insult. As an alternative to determining the normalcytokine level for the particular subject, the normal level can beequated to the average value obtained or known for a group of similarlysituated healthy individuals (i.e., a group of healthy individualshaving the same or similar physical condition—age, gender, weight, diet,etc.—and medical history). This alternative approach may be necessarywhen the normal cytokine level for the given subject cannot bepre-determined (e.g., the individual has been subjected to an unforeseeninsult such as a physical injury resulting from an accident) and is nototherwise available in the subject's medical history. Cytokine levelsare typically determined in vitro using a sample of the subject's blood.Cytokine levels can be determined, for example, in accordance with themethods described in Casey et al., Ann. Intern. Med. 1993, 119(8):771-778, and in Bolke et al. Shock 2001, 16(5): 334-9.

A proinflammatory property of IL6 is its ability to stimulateprostaglandin synthesis. Impaired febrile responses are evident in micewhich lack the prostaglandin E2 receptor subtype EP3. Consequently, theefficacy of the administration of a CCR5 antagonist in treating orpreventing a stress response (e.g., a post-surgical febrile response)can be determined by monitoring the level of production of prostaglandinE2 (PGE2). Accordingly, the present invention also includes a method fortreating or preventing stress response (including, e.g., febrileresponse) in a subject in need thereof, which comprises administering tothe subject a CCR5 antagonist in an amount effective to inhibitendogenous production of prostaglandin E2. PGE2 levels can be measuredin accordance with the method described in Brideau et al., Inflamm. Res.1996, (45): 68-74.

In the present invention, the chemokine modulators (e.g., CCR5antagonists) can be administered with one or more otheranti-inflammatory agents, including blockers of other chemokine receptorpathways, steroids, and non-steroidal anti-inflammatory agents. Thisapproach can be viewed as an alternative to anti-TNF, solubleTNF-receptor compounds, or other anti-cytokine agents (IL1, IL6, etc.),none of which alone are believed to provide the effect observed inaccordance with the present invention. When the chemokine modulator isadministered with another agent (i.e., co-administration), it isunderstood that the modulator can be administered before, concurrentlywith, or after administration of the other agent. When administeredconcurrently, the modulator and the agent can be administered separatelyat the same time or together in one composition.

Chemokine receptor CCR5 modulators are used in the present methods formodulating chemokine receptor CCR5 activity in tissues, includingmodulating stress responses in tissues. Thus, as used herein, the terms“modulate”, “modulating”, and “modulator” are meant to be construed toencompass inhibiting, blocking, promoting, stimulating, agonizing,antagonizing, or otherwise affecting chemokine receptor CCR5 activity intissues.

Such modulators can take a variety of forms that include compounds thatinteract with the chemokine receptor CCR5 in a manner such thatfunctional interactions with natural chemokine receptor ligands aremimicked, stimulated and/or inhibited. Exemplary modulators includeanalogs of a chemokine receptor natural ligand binding site on achemokine receptor CCR5, mimetics of a natural ligand of a chemokinereceptor that mimic the structural region involved in chemokinereceptor-receptor ligand binding interactions, polypeptides having asequence corresponding to the domain of a natural ligand of a chemokinereceptor, and antibodies which immunoreact with either a chemokinereceptor or the natural ligand, all of which exhibit modulator activityas defined herein.

Small organic molecules which are chemokine receptor CCR5 modulators,especially CCR5 antagonists, are suitable for use in the methods of thepresent invention. Thus, the present invention includes a method oftreating or preventing stress response which comprises administering toa subject in need of such treatment a therapeutically effective amountof a compound of Formula (I), or a pharmaceutically acceptable salt oran individual diastereomer thereof:

wherein:

-   -   X is selected from: —(C₀₋₆ alkyl)-Y—(C₀₋₆ alkyl)-,        -   —(C₀₋₆ alkyl)-C₃₋₈ cycloalkyl-(C₀₋₆ alkyl)-,        -   C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl,        -   where the alkyl is unsubstituted or substituted with 1-7            substituents where the substituents are independently            selected from:        -   (a) halo,        -   (b) hydroxy,        -   (c) —O—C₁₋₃ alkyl, and        -   (d) trifluoromethyl,    -   and where Y is selected from:        -   a single bond, —O—, —SO₂—, —NR¹⁰—, —NR¹⁰—SO₂—, —SO₂—NR¹⁰—,            —S—, and —SO—,    -   and where R¹⁰ is independently selected from: hydrogen, C₁₋₆        alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₅₋₆ cycloalkyl, benzyl,        phenyl, and C₁₋₆ alkyl-C₃₋₆ cycloalkyl,        -   which is unsubstituted or substituted with 1-3 substituents            where the substituents are independently selected from:            halo, C₁₋₃ alkyl, C₁₋₃ alkoxy and trifluoromethyl;    -   R¹ is selected from:        -   (1) —CO₂H,        -   (2) —NO₂,        -   (3) -tetrazolyl,        -   (4) -hydroxyisoxazole,        -   (5) —SO₂NHCO—(C₀₋₃ alkyl)-R⁹, wherein R⁹ is independently            selected from: hydrogen, C₁₋₆ alkyl, C₅₋₆ cycloalkyl, benzyl            or phenyl, which is unsubstituted or substituted with 1-3            substituents where the substituents are independently            selected from: halo, C₁₋₃ alkyl, C₁₋₃ alkoxy and            trifluoromethyl, and        -   (6) —P(O)(OH)₂;    -   R³ is selected from the group consisting of:        -   phenyl and heterocycle,            -   which is unsubstituted or substituted with 1-7                substituents where the substituents are independently                selected from:            -   (a) halo,            -   (b) trifluoromethyl,            -   (c) hydroxy,            -   (d) C₁₋₃ alkyl,            -   (e) —O—C₁₋₃ alkyl,            -   (f) —CO₂R⁹,            -   (g) —NR⁹R¹⁰, and            -   (h) —CONR⁹R¹⁰;    -   R⁴, R⁵ and R⁶ are independently selected from:        -   hydrogen, C₁₋₁₀ alkyl, C₃₋₈ cycloalkyl, C₂₋₁₀ alkenyl,        -   C₂₋₁₀ alkynyl, phenyl, —(C₁₋₆ alkyl)-phenyl,        -   —(C₁₋₆ alkyl)-C₃₋₈ cycloalkyl, naphthyl, biphenyl, and            heterocycle,            -   which is unsubstituted or substituted with 1-7 of R¹¹                where R¹¹ is independently selected from:            -   (a) halo,            -   (b) trifluoromethyl,            -   (c) hydroxy,            -   (d) C₁₋₃ alkyl,            -   (e) —O—C₁₋₃ alkyl,            -   (f) —CO₂R⁹,            -   (g) —NR⁹R¹⁰, and            -   (h) —CONR⁹R¹⁰,    -   or where R⁴ and R⁵ may be joined together to form a 3-8 membered        saturated ring which may be unsubstituted or substituted with        1-7 of R¹¹,    -   or where R⁵ and R⁶ may be joined together to form a 3-8 membered        saturated ring which may be unsubstituted or substituted with        1-7 of R¹¹;    -   R⁷ is selected from:        -   (1) hydrogen,        -   (2) C₁₋₆ alkyl, which is unsubstituted or substituted with            1-4 substituents where the substituents are independently            selected from: hydroxy, cyano, and halo,        -   (3) hydroxy, and        -   (4) halo;    -   R⁸ is selected from:        -   hydrogen, C₃₋₈ cycloalkyl, phenyl, naphthyl, biphenyl, and            heterocycle,            -   which is unsubstituted or substituted with 1-7 of R¹²                where R¹² is independently selected from:            -   (a) halo,            -   (b) cyano,            -   (c) hydroxy,            -   (d) C₁₋₆ alkyl, which is unsubstituted or substituted                with 1-5 of R¹³ where R¹³ is independently selected                from: halo, cyano, hydroxy, C₁₋₆ alkoxy, —CO₂H,                —CO₂(C₁₋₆ alkyl), trifluoromethyl, and —NR⁹R¹⁰,            -   (e) —O—C₁₋₆ alkyl, which is unsubstituted or substituted                with 1-5 of R¹³,            -   (f) —CF₃,            -   (g) —CHF₂,            -   (h) —CH₂F,            -   (i) —NO₂,            -   (j) C₀₋₆ alkyl-phenyl or C₀₋₆ alkyl-heterocycle, which                is unsubstituted or substituted with 1-7 substituents                where the substituents are independently selected from:                -   (i) halo,                -   (ii) hydroxy,                -   (iii) C₁₋₆ alkyl, unsubstituted or substituted with                    1-5 substituents, each of which is independently                    selected from halo, cyano, hydroxy, C₁₋₆ alkoxy,                    —CO₂H, —CO₂(C₁₋₆ alkyl), trifluoromethyl, and                    —NR⁹R¹⁰,                -   (iv) —O—C₁₋₆ alkyl,                -   (v) —CF₃,                -   (vi) —OCF₃,                -   (vii) —NO₂,                -   (viii) —CN,                -   (ix) —SO₂—C₁₋₆ alkyl,                -   (x) —CO₂R⁹,                -   (xi) —NR⁹R¹⁰,                -   (xii) —CONR⁹R¹⁰,                -   (xiii) —SO₂—NR⁹R¹⁰,                -   (xiv) —NR⁹—SO₂—R¹⁰,                -   (xv) —C₃₋₈ cycloalkyl,                -   (xvi) —OC₃₋₈ cycloalkyl, and                -   (xvii) phenyl;            -   (k) —CO₂R⁹,            -   (l) tetrazolyl,            -   (m) —NR⁹R¹⁰,            -   (n) —NR⁹—COR¹⁰,            -   (o) —NR⁹—CO₂R¹⁰,            -   (p) —CO—NR⁹R¹⁰,            -   (q) —OCO—NR⁹R¹⁰,            -   (r) —NR⁹CO—NR⁹R¹⁰,            -   (s) —S(O)_(m)—R⁹,wherein m is an integer selected from                0, 1 and 2,            -   (t) —S(O)₂—NR⁹R¹⁰,            -   (u) —NR⁹S(O)₂—R¹⁰,            -   (v) —NR⁹S(O)₂—NR⁹R¹⁰,            -   (w) C₁₋₆ alkyl substituted with —C₃₋₈ cycloalkyl, and            -   (x) —C₃₋₈ cycloalkyl;    -   n is an integer selected from 1, 2, 3 and 4;    -   x is an integer selected from 0, 1 and 2, and y is an integer        selected from 0, 1 and 2,    -   with the proviso that the sum of x and y is 2.

Embodiments of the preceding method include the method as just describedincorporating one or more of the following features:

-   -   (1a) R¹ in the compound of Formula (I) is selected from —CO₂H        and -tetrazolyl.    -   (1b) R¹ in the compound of Formula (I) is —CO₂H.    -   (2a) R³ in the compound of Formula (I) is selected from the        group consisting of phenyl and thienyl, which may be        unsubstituted or substituted with 1-5 substituents where the        substituents are independently selected from:        -   (a) halo,        -   (b) trifluoromethyl,        -   (c) hydroxy        -   (d) C₁₋₃ alkyl, and        -   (e) —O—C₁₋₃ alkyl.    -   (2b) R³ in the compound of Formula (I) is selected from the        group consisting of phenyl, which may be unsubstituted or        substituted with 1-5 substituents where the substituents are        independently selected from (a) fluoro, and (b) chloro; and        unsubstituted thienyl.    -   (2c) R³ in the compound of Formula (I) is unsubstituted phenyl,        (3-fluoro)phenyl or 3-thienyl.    -   (3) R⁴ in the compound of Formula (I) is hydrogen.    -   (4a) R⁵ in the compound of Formula (I) is selected from        hydrogen, C₁₋₆ alkyl, C₃₋₈ cycloalkyl, C₁₋₆ alkyl-C₃₋₈        cycloalkyl, and phenyl.    -   (4b) R⁵ in the compound of Formula (I) is selected from        hydrogen, methyl, n-propyl, isopropyl, n-butyl, t-butyl,        isobutyl, sec-butyl, cyclohexyl, —CH₂-cyclopropyl,        —CH₂-cyclobutyl, and phenyl.    -   (4c) R⁵ in the compound of Formula (I) is selected from        isopropyl, isobutyl, sec-butyl, and cyclohexyl.    -   (5a) R⁶ in the compound of Formula (I) is selected from        hydrogen, C₁₋₆ alkyl, C₃₋₈ cycloalkyl, C₁₋₆ alkyl-C₃₋₈        cycloalkyl, and phenyl.    -   (5b) R⁶ in the compound of Formula (I) is selected from        hydrogen, methyl, n-butyl, t-butyl, isobutyl, sec-butyl,        —CH₂-cyclopropyl, —CH₂-cyclobutyl, and cyclohexyl.    -   (5c) R⁶ in the compound of Formula (I) is selected from        hydrogen, methyl, —CH₂-cyclopropyl, —CH₂-cyclobutyl, and        cyclohexyl.    -   (6a) R⁷ in the compound of Formula (I) is hydrogen, fluoro,        hydroxy or C₁₋₆ alkyl.    -   (6b) R⁷ in the compound of Formula (I) is hydrogen.    -   (7a) X in the compound of Formula (I) is: —(C₀₋₄ alkyl)-Y—(C₀₋₄        alkyl)-, where the alkyl is unsubstituted or substituted with        1-4 substituents where the substituents are independently        selected from:        -   (a) halo,        -   (b) hydroxy,        -   (c) —O—C₁₋₃ alkyl, and        -   (d) trifluoromethyl,            and where Y is selected from:    -   a single bond, —O—, —SO₂—, —NR¹⁰—, —S—, and —SO—,        and where R¹⁰ is independently selected from: hydrogen, C₁₋₆        alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, benzyl, phenyl, and C₁₋₆        alkyl-C₃₋₆ cycloalkyl, which is unsubstituted or substituted        with 1-3 substituents where the substituents are independently        selected from: halo, C₁₋₃ alkyl, C₁₋₃ alkoxy and        trifluoromethyl.    -   (7b) X in the compound of Formula (1) is: —(C₀₋₂ alkyl)-Y—(C₀₋₂        alkyl)-, where the alkyl is unsubstituted or substituted with        1-4 substituents where the substituents are independently        selected from:        -   (a) halo,        -   (b) hydroxy,        -   (c) —O—C₁₋₃ alkyl, and        -   (d) trifluoromethyl,            and where Y is selected from:    -   a single bond, —O—, —SO₂—, —NR¹⁰—, —S—, and —SO—,        where R¹⁰ is independently selected from: hydrogen, C₁₋₆ alkyl,        C₂₋₆ alkenyl, C₂₋₆ alkynyl, benzyl, phenyl, and C₁₋₆ alkyl-C₃₋₆        cycloalkyl,    -   which is unsubstituted or substituted with 1-3 substituents        where the substituents are independently selected from: halo,        C₁₋₃ alkyl,    -   C₁₋₃ alkoxy and trifluoromethyl.    -   (7c) X in the compound of Formula (I) is selected from —(C₀₋₂        alkyl)-Y—(C₀₋₂ alkyl)-, where the alkyl is unsubstituted or        substituted with fluoro,        and where Y is selected from:    -   a single bond, —SO₂—, —SO—, and —NR¹⁰—,        where R¹⁰ is independently selected from: hydrogen, C₁₋₃ alkyl,        C₂₋₃ alkenyl, and C₂₋₃ alkynyl.    -   (7d) X in the compound of Formula (I) is selected from:        -   (1) a single bond,        -   (2) —CH₂CH₂—,        -   (3) —CH₂CH₂CH₂—,        -   (4) —CH₂CH₂—CF₂—,        -   (5) —CH₂CH₂—SO₂—, and        -   (6) —CH₂CH₂—SO—.    -   (8a) R⁸ in the compound of Formula (I) is selected from: phenyl,        naphthyl, cyclohexyl, benzoimidazolyl, benzofurazanyl,        imidazopyridyl, imidazolyl, isoxazolyl, oxazolyl, pyrazinyl,        pyridazinyl, pyridyl, pyrimidyl, thiazolyl, tetrazolopyridyl,        and pyrazolyl;        -   which is unsubstituted or substituted with 1-7 substituents            where the substituents are independently selected from:            -   (a) halo,            -   (b) cyano,            -   (c) hydroxy,            -   (d) C₁₋₆ alkyl, which is unsubstituted or substituted                with 1-5 of R¹³ where R¹³ is independently selected                from: halo, cyano, hydroxy, C₁₋₆ alkoxy, —CO₂H,                —CO₂(C₁₋₆ alkyl), trifluoromethyl, and —NR⁹R¹⁰, wherein                R⁹ and R¹⁰ are independently selected from: hydrogen,                C₁₋₆ alkyl, C₅₋₆ cycloalkyl, benzyl or phenyl, which is                unsubstituted or substituted with 1-3 substituents where                the substituents are independently selected from: halo,                C₁₋₃ alkyl, C₁₋₃ alkoxy and trifluoromethyl;            -   (e) —O—C₁₋₆ alkyl, which is unsubstituted or substituted                with 1-5 of R¹³,            -   (f) —CF₃,            -   (g) —CHF₂,            -   (h) —CH₂F,            -   (i) —NO₂,            -   (j) C₀₋₆ alkyl-phenyl or C₀₋₆ alkyl-heterocycle, which                is unsubstituted or substituted with 1-7 substituents                where the substituents are independently selected from:                -   (i) halo,                -   (ii) hydroxy,                -   (iii) C₁₋₆ alkyl,                -   (iv) —O—C₁₋₆ alkyl,                -   (v) —CF₃,                -   (vi) —OCF₃,                -   (vii) —NO₂,                -   (viii) —CN,                -   (ix) —SO₂—C₁₋₆ alkyl,                -   (x) —CO₂R⁹,                -   (xi) —NR⁹R¹⁰,                -   (xii) —CONR⁹R¹⁰,                -   (xiii) —SO₂—NR⁹R¹⁰, and                -   (xiv) —NR⁹—SO₂—R¹⁰;            -   (k) —CO₂R⁹,            -   (l) tetrazolyl,            -   (m) —NR⁹R¹⁰,            -   (n) —NR⁹—COR¹⁰,            -   (o) —NR⁹—CO₂R¹⁰,            -   (p) —CO—NR⁹R¹⁰,            -   (q) —OCO—NR⁹R¹⁰,            -   (r) —NR⁹CO—NR⁹R¹⁰,            -   (s) —S(O)_(m)—R⁹, wherein m is an integer selected from                0, 1 and 2,            -   (t) —S(O)₂—NR⁹R¹⁰,            -   (u) —NR⁹S(O)₂—R¹⁰, and            -   (v) —NR⁹S(O)₂—NR⁹R¹⁰.    -   (8b) R⁸ in the compound of Formula (I) is selected from phenyl,        imidazopyridyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, and        thiazolyl;        -   which is unsubstituted or substituted with 1-5 substituents            where the substituents are independently selected from:        -   (a) halo,        -   (b) cyano,        -   (c) —NO₂,        -   (d) —CF₃,        -   (e) —CHF₂,        -   (f) —CH₂F,        -   (h) C₁₋₆ alkyl,        -   (i) C₁₋₃ alkyl-phenyl or C₁₋₃ alkyl-pyridyl, which is            unsubstituted or substituted with 1-4 substituents where the            substituents are independently selected from:            -   (i) halo,            -   (ii) C₁₋₆ alkyl,            -   (iii) —O—C₁₋₆ alkyl,            -   (iv) —CF₃,            -   (vi) —OCF₃,            -   (vii) —CN, and        -   (j) —O—C₁₋₆ alkyl.    -   (8c) R⁸ in the compound of Formula (I) is selected from        imidazolyl, oxazolyl, pyrazolyl, and thiazolyl; which is        unsubstituted or substituted with 1-3 substituents where the        substituents are independently selected from:        -   (a) fluoro,        -   (b) cyano,        -   (c) C₁₋₃ alkyl,        -   (d) —CH₂-phenyl, which is unsubstituted or substituted with            1-4 substituents where the substituents are independently            selected from:            -   (i) fluoro,            -   (ii) chloro,            -   (iii) —O—CH₃,            -   (iv) —CF₃,            -   (v) —CN, and    -   (e) —CF₃.    -   (8d) R⁸ in the compound of Formula (I) is selected from        5-(3-benzyl)pyrazolyl, 5-(1-methyl-3-benzyl)pyrazolyl,        5-(1-ethyl-3-benzyl)pyrazolyl, 5-(2-benzyl)thiazolyl,        5-(2-benzyl-4-methyl)thiazolyl, and        5-(2-benzyl-4-ethyl)thiazolyl).    -   (9) n in the compound of Formula (I) is an integer which is 1.    -   (10) In the compound of Formula (I), x is an integer which is 1        and y is an integer which is 1.    -   (11) The subject is a warm-blooded vertebrate.    -   (12) The subject is a primate.    -   (13) The subject is a human.    -   (14) The subject is other than a graft transplant patient.    -   (15) The subject is a cardiac surgery patient.    -   (16) The stress response comprises hyperthermia.    -   (17) The stress response comprises a response to surgery (e.g.,        surgical hyperthermia).    -   (18) The stress response comprises hypothermia (e.g., surgical        hypothermia).

The present invention also includes a method of treating or preventingstress response in a subject in need thereof, which comprisesadministering to the subject a compound of Formula (I), or apharmaceutically acceptable salt or an individual diastereomer thereof,in an amount effective to inhibit endogenous production of one or morepro-inflammatory cytokines selected from the group consisting of IL1,IL6, and TNF (e.g., one or more cytokines selected from the groupconsisting of IL1 and IL6). Embodiments of this method include themethod as just described incorporating one or more of the features (1)to (18) as set forth above for the preceding method directed to CompoundI.

The present invention further includes a method of treating orpreventing stress response which comprises administering to a subject inneed thereof a therapeutically effective amount of a compound of Formula(II), or a pharmaceutically acceptable salt thereof:

wherein

-   -   R⁴is    -   R⁸ is selected from the group consisting of    -   R¹² and R¹⁴ are each independently selected from the group        consisting of F, Cl, CF₃, OCH₃, OCH₂CH₃, OCF₃, O-cyclobutyl, CN,        O-cyclopropyl, CH₃, CH₂CH₃, CH(CH₃)₂, C(CH₃)₃, and SO₂CH₃;    -   G is hydrogen or fluoro; and    -   q is an integer equal to 1 or 2.

Embodiments of the preceding method include the method as justdescribed, except that the stress response comprises hyperthermia, orthe stress response comprises a response to surgery, or the stressresponse comprises hypothermia. Aspects of the method as first describedand of the immediately preceding embodiments include the methods inwhich the subject is a warm-blooded vertebrate, or is a primate, or is ahuman, or is other than a graft transplant patient, or is a cardiacsurgery patient.

The present invention also includes a method of treating or preventingstress response in a subject in need thereof, which comprisesadministering to the subject a compound of Formula (II), or apharmaceutically acceptable salt thereof, in an amount effective toinhibit endogenous production of one or more pro-inflammatory cytokinesselected from the group consisting of IL1, IL6, and TNF (e.g., one ormore cytokines selected from the group consisting of IL1 and IL6).Embodiments of this method include the method as just describedincorporating one or more of the embodiments or aspects as set forth inthe preceding paragraph directed to related methods involving CompoundII.

The present invention also includes a method of treating stress responsewhich comprises administering to a subject in need of such treatment atherapeutically effective amount of Compound A:

or a pharmaceutically acceptable salt thereof. Embodiments of thismethod include the method as just described, except that the stressresponse comprises hyperthermia, or the stress response comprises aresponse to surgery, or the stress response comprises hypothermia.Aspects of the method as first described and of the immediatelypreceding embodiments include the methods in which the subject is awarm-blooded vertebrate, or is a primate, or is a human, or is otherthan a graft transplant patient, or is a cardiac surgery patient.

The present invention also includes a method of treating or preventingstress response in a subject in need thereof, which comprisesadministering to the subject Compound A, or a pharmaceuticallyacceptable salt thereof, in an amount effective to inhibit endogenousproduction of one or more pro-inflammatory cytokines selected from thegroup consisting of IL1, IL6, and TNF (e.g., one or more cytokinesselected from the group consisting of IL1 and IL6). Embodiments of thismethod include the method as just described incorporating one or more ofthe embodiments or aspects as set forth in the preceding paragraphdirected to related methods involving Compound A.

Compounds of Formula (I), compounds of Formula (II), and Compound A canbe prepared as described in U.S. Pat. No. 6,358,979, which is based onU.S. Ser. No. 09/590,750, filed Jun. 8, 2000; and in WO 00/76972, thedisclosures of which are herein incorporated by reference in theirentireties. These compounds have exhibited activity in binding to theCCR5 receptor, as described in U.S. Pat. No. 6,358,979 and WO 00/76972.

As indicated above, small molecule organic chemokine receptor CCR5modulators suitable for use in the present invention can be administeredin the form of pharmaceutically acceptable salts. The term“pharmaceutically acceptable salt” refers to a salt which possesses theeffectiveness of the parent compound and which is not biologically orotherwise undesirable (e.g., is neither toxic nor otherwise deleteriousto the recipient thereof). Suitable salts include acid addition saltswhich may, for example, be formed by mixing a solution of the compoundof the present invention with a solution of a pharmaceuticallyacceptable acid such as hydrochloric acid, sulfuric acid, acetic acid,trifluoroacetic acid, or benzoic acid. When the compounds of theinvention carry an acidic moiety, suitable pharmaceutically acceptablesalts thereof can include alkali metal salts (e.g., sodium or potassiumsalts), alkaline earth metal salts (e.g., calcium or magnesium salts),and salts formed with suitable organic ligands such as quaternaryammonium salts. Also, in the case of an acid (—COOH) or alcohol groupbeing present, pharmaceutically acceptable esters can be employed tomodify the solubility or hydrolysis characteristics of the compound.

Other small molecule organic chemokine receptor CCR5 modulators(especially CCR5 antagonists) suitable for use in the present inventioninclude, for example, those described in U.S. Pat. No. 6,013,644, U.S.Pat. No. 5,962,462, U.S. Pat. No. 5,919,776, U.S. Pat. No. 6,124,319,U.S. Pat. No. 6,136,827, U.S. Pat. No. 6,166,037, U.S. Pat. No.6,140,349, U.S. Pat. No. 6,265,434, U.S. Pat. No. 6,248,755, WO00/59498, WO 00/59497, WO 99/76512, WO 00/76511, WO 00/76973, WO00/76513, and WO 00/76514.

Polypeptides are also suitable for use as chemokine modulators in thepresent invention. A polypeptide (peptide) modulator interacts with thechemokine receptor CCR5 and can correspond in sequence to a naturalligand. As used herein, the term “polypeptide” refers to a linear orcyclic compound comprising from about 2 to no more than about 100 aminoacid residues, wherein the amino group of one amino acid is linked tothe carboxyl group of another amino acid by a peptide bond. In oneembodiment, polypeptides containing from about 2 to about about 60residues are employed in the present invention. In another embodiment,polypeptides containing from about 2 to about 30 residues are employed.It is understood that a suitable polypeptide need not be identical tothe amino acid residue sequence of a natural ligand, so long as itincludes required binding sequences and is able to function as achemokine receptor CCR5 modulator, especially a CCR5 antagonist.

A suitable polypeptide includes any analog, fragment or chemicalderivative of a polypeptide that is a chemokine receptor CCR5 modulator.Such a polypeptide can be subject to various changes, substitutions,insertions, and deletions where such changes provide for certainadvantages in its use (e.g., improvement in the potency of thepolypeptide or conversion of the polypeptide from an agonist to anantagonist of the CCR5 receptor). A modulator polypeptide suitable foruse in the present invention can correspond to, rather than be identicalto, the sequence of a natural ligand where one or more changes are madein the sequence and it retains the ability to function as a chemokinereceptor CCR5 modulator. Thus, a suitable polypeptide can be in any of avariety of forms of peptide derivatives, that include amides, conjugateswith proteins, cyclized peptides, polymerized peptides, analogs,fragments, chemically modified peptides, and the like, provided it is amodulator of chemokine receptor CCR5 activity.

The “analog” of a polypeptide refers to any polypeptide having an aminoacid residue sequence substantially identical to a sequence of a naturalligand of a chemokine receptor CCR5 in which one or more residues havebeen conservatively substituted with a functionally similar residue andwhich displays the requisite chemokine receptor modulator activity.Examples of conservative substitutions include the substitution of onenon-polar (hydrophobic) residue such as isoleucine, valine, leucine ormethionine for another; the substitution of one polar (hydrophilic)residue for another such as between arginine and lysine, betweenglutamine and asparagine, between glycine and serine; the substitutionof one basic residue such as lysine, arginine or histidine for another;or the substitution of one acidic residue, such as aspartic acid orglutamic acid for another. The phrase “conservative substitution” alsoincludes the use of a chemically derivatized residue in place of anon-derivatized residue provided that such polypeptide displays therequisite inhibition activity. The phrase “chemical derivatizedpolypeptide” refers to a subject polypeptide having one or more residueschemically derivatized by reaction of a functional side group. Suchderivatized molecules include, for example, those molecules in whichfree amino groups have been derivatized to form amine hydrochlorides,p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonylgroups, chloroacetyl groups or formyl groups. Free carboxyl groups canbe derivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups can be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine canbe derivatized to form N-imbenzylhistidine. Also included as chemicalderivatives are those peptides that contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexample, 4-hydroxyproline can be substituted for proline;5-hydroxylysine can be substituted for lysine; 3-methylhistidine can besubstituted for histidine; homoserine can be substituted for serine; andornithine can be substituted for lysine.

Additional residues can also be added at either terminus of apolypeptide for the purpose of providing a “linker” by which thepolypeptides of the present invention can be conveniently affixed to alabel or solid matrix, or carrier. Labels, solid matrices and carriersthat can be used with the polypeptides of the present invention aredescribed hereinbelow. Amino acid residue linkers are usually at leastone residue and can be 40 or more residues, more often 1 to 10 residues,but do not form chemokine receptor ligand epitopes. Typical amino acidresidues used for linking are tyrosine, cysteine, lysine, glutamic andaspartic acid, or the like. In addition, a subject polypeptide candiffer, unless otherwise specified, from the natural sequence of aligand by the sequence being modified by terminal-NH2 acylation, e.g.,acetylation, or thioglycolic acid amidation, byterminal-carboxylamidation, e.g., with ammonia, methylamine, and thelike terminal modifications. Terminal modifications are useful, as iswell known, to reduce susceptibility by proteinase digestion, andtherefore serve to prolong half life of the polypeptides in solutions,particularly biological fluids where proteases can be present. In thisregard, polypeptide cyclization is also a useful terminal modification,and is particularly preferred also because of the stable structuresformed by cyclization.

Any polypeptide suitable for use in the present invention can beemployed in the form of a pharmaceutically acceptable salt. Suitableacids which are capable of forming salts with the peptides includeinorganic acids such as trifluoroacetic acid (TFA), hydrochloric acid(HCl), hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,sulfuric acid, phosphoric acetic acid, propionic acid, glycolic acid,lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid, sulfanilic acid or the like. Suitable bases capable offorming salts with the peptides of the present invention includeinorganic bases such as sodium hydroxide, ammonium hydroxide, potassiumhydroxide and the like; and organic bases such as mono-di- and tri-alkyland aryl amines (e.g. triethylamine, diisopropyl amine, methyl amine,dimethyl amine and the like), and optionally substituted ethanolamines(e.g. ethanolamine, diethanolamine and the like).

A polypeptide suitable for use in the present invention can besynthesized by techniques known to those skilled in the polypeptide art,including recombinant DNA techniques. Synthetic chemistry techniques,such as a solid-phase Merrifield-type synthesis, are preferred forreasons of purity, antigenic specificity, freedom from undesired sideproducts, ease of production and the like. Suitable techniques forpreparing polypeptides include those described in Steward et al., “SolidPhase Peptide Synthesis”, W. H. Freeman Co., San Francisco, 1969;Bodanszky et al., “Peptide Synthesis”, John Wiley & Sons, SecondEdition, 1976; J. Meienhofer, “Hormonal Proteins and Peptides”, Vol. 2,p. 46, Academic Press (New York), 1983; Merrifield, Adv. Enzymol. 1969,32: 221-96; Fields et al., Int. J. Peptide Protein Res. 1990, 35:161-214; U.S. Pat. No. 4,244,946 for solid phase peptide synthesis; andSchroder et al., “The Peptides”, Vol. 1, Academic Press (New York), 1965for classical solution synthesis; each of which is incorporated hereinby reference in its entirety. Appropriate protective groups usable insuch syntheses are described in the above texts and in J. F. W. McOmie,“Protective Groups in Organic Chemistry”, Plenum Press, New York, 1973,which is incorporated herein by reference in its entirety, and in T. W.Greene and P. G. M. Wuts, “Protective Groups in Organic Synthesis”,2^(nd) edition, John Wiley & Sons, New York, 1991, which is incorporatedherein by reference in its entirety.

In general, the solid-phase synthesis methods comprise the sequentialaddition of one or more amino acid residues or suitably protected aminoacid residues to a growing peptide chain. Normally, either the amino orcarboxyl group of the first amino acid residue is protected by asuitable, selectively removable protecting group. A different,selectively removable protecting group is utilized for amino acidscontaining a reactive side group such as lysine.

In a representative solid phase synthesis, the protected or derivatizedamino acid is attached to an inert solid support through its unprotectedcarboxyl or amino group. The protecting group of the amino or carboxylgroup is then selectively removed and the next amino acid in thesequence having the complementary (amino or carboxyl) group suitablyprotected is admixed and reacted under conditions suitable for formingthe amide linkage with the residue already attached to the solidsupport. The protecting group of the amino or carboxyl group is thenremoved from this newly added amino acid residue, and the next aminoacid (suitably protected) is then added, and so forth. After all thedesired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to afford the final linearpolypeptide.

Linear polypeptides, such as a linear peptide prepared by a solid phasesynthesis as just described, can be reacted to form their correspondingcyclic peptides. An exemplary method for cyclizing peptides is describedon pages 393-394 of Zimmer et al., “Peptides 1992”, ESCOM SciencePublishers, B. V., 1993, which is herein incorporated by reference inits entirety. Typically, tert-butoxycarbonyl protected peptide methylester is dissolved in methanol, sodium hydroxide solution is added, andthe admixture is reacted at 20° C. to hydrolytically remove the methylester protecting group. After evaporating the solvent, thetert-butoxycarbonyl protected peptide is extracted with ethyl acetatefrom acidified aqueous solvent. The tert-butoxycarbonyl protecting groupis then removed under mildly acidic conditions in dioxane cosolvent. Theunprotected linear peptide with free amino and carboxy termini soobtained is converted to its corresponding cyclic peptide by reacting adilute solution of the linear peptide, in a mixture of dichloromethaneand dimethylformamide, with dicyclohexylcarbodiimide in the presence of1-hydroxybenzotriazole and N-methylmorpholine. The resultant cyclicpeptide is then purified by chromatography.

Antibodies are also suitable for use as chemokine receptor CCR5modulators in the present invention, wherein the antibodies, includingmonoclonal antibodies, immunoreact with a chemokine receptor CCR5 and/orbind the chemokine receptor to modulate receptor activity. The term“antibody”, or a variant thereof (e.g., “antibody molecule”), refers toa population of immunoglobulin molecules and/or immunologically activeportions of immunoglobulin molecules; i.e., molecules that contain anantibody combining site or paratope. An “antibody combining site” isthat structural portion of an antibody molecule comprised of heavy andlight chain variable and hypervariable regions that specifically bindsantigen.

Exemplary antibodies suitable for use in the present invention areintact immunoglobulin molecules, substantially intact immunoglobulinmolecules (i.e., immunoglobulins having changes in sequence that do notaffect its ability to fix complement or to interact with Fc receptors),single chain immunoglobulins or antibodies, those portions of animmunoglobulin molecule that contain the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′)2 and F(v), and alsoreferred to as antibody fragments. The phrase “monoclonal antibody”, ora variant thereof, refers to a population of antibody molecules thatcontain only one species of antibody combining site capable ofimmunoreacting with a particular epitope. A monoclonal antibody thustypically displays a single binding affinity for any epitope with whichit immunoreacts. A monoclonal antibody can therefore contain an antibodymolecule having a plurality of antibody combining sites, eachimmunospecific for a different epitope, e.g., a bispecific monoclonalantibody.

A monoclonal antibody is typically composed of antibodies produced byclones of a single cell called a hybridoma that secretes (produces) onlyone kind of antibody molecule. The hybridoma cell is formed by fusing anantibody-producing cell and a myeloma or other self-perpetuating cellline. The preparation of such antibodies was first described by Kohlerand Milstein, Nature 1975, 256: 495-497, which description isincorporated by reference in its entirety. Additional methods aredescribed by Zola, “Monoclonal Antibodies: a Manual of Techniques”, CRCPress, Inc., 1987. The hybridoma supernates so prepared can be screenedfor the presence of antibody molecules that immunoreact with a chemokinereceptor and modulate its biological function.

Briefly, to form the hybridoma from which the monoclonal antibodycomposition is produced, a myeloma or other self-perpetuating cell lineis fused with lymphocytes obtained from the spleen of a mammalhyperimmunized with a source of a chemokine receptor, as described byCheresh et al., J. Biol. Chem. 1987, 262: 17703-17711, hereinincorporated by reference in its entirety. It is preferred that themyeloma cell line used to prepare a hybridoma be from the same speciesas the lymphocytes. Typically, a mouse of the strain 129 G1X+ is thepreferred mammal. Suitable mouse myelomas include thehypoxanthine-aminopterin-thymidine-sensitive (HAT) cell linesP3X63-Ag8.653, and Sp2/0-Ag14 that are available from the ATCC,Manassas, Va., under the designations CRL 1580 and CRL 1581,respectively. Splenocytes are typically fused with myeloma cells usingpolyethylene glycol (PEG) 1500. Fused hybrids are selected by theirsensitivity to HAT. Hybridomas producing a monoclonal antibody of thepresent invention can be identified using the enzyme linkedimmunosorbent assay.

A suitable monoclonal antibody can also be produced by initiating amonoclonal hybridoma culture comprising a nutrient medium containing ahybridoma that secretes antibody molecules of the appropriatespecificity. The culture is maintained under conditions and for a timeperiod sufficient for the hybridoma to secrete the antibody moleculesinto the medium. The antibody-containing medium is then collected. Theantibody molecules can then be further isolated by well knowntechniques. Media useful for the preparation of these compositions areboth well known in the art and commercially available and includesynthetic culture media, inbred mice and the like. An exemplarysynthetic medium is Dulbecco's minimal essential medium (DMEM—Dulbeccoet al., Virol. 1959, 8: 396) supplemented with 4.5 gm/1 glucose, 20 mMglutamine, and 20% fetal calf serum. An exemplary inbred mouse strain isthe Balb/C.

Other methods of producing a monoclonal antibody, a hybridoma cell, or ahybridoma cell culture include, for example, the method of isolatingmonoclonal antibodies from an immunological repertoire as described bySastry, et al., Proc Natl. Acad. Sci. USA 1989, 86: 5728-5732; and Huseet al., Science 1989, 246: 1275-1281, each of which is hereinincorporated by reference in its entirety. Also suitable for use in thepresent invention are monoclonal antibodies produced from culturescontaining a hybridoma cell.

It is also possible to determine, without undue experimentation, if amonoclonal antibody has the same (i.e., equivalent) specificity(immunoreaction characteristics) as a monoclonal antibody suitable foruse in the present invention by ascertaining whether the former preventsthe latter from binding to a preselected target molecule. If themonoclonal antibody being tested competes with the monoclonal antibodyof the invention, as shown by a decrease in binding by the monoclonalantibody of the invention in standard competition assays for binding tothe target molecule when present in the solid phase, then it is likelythat the two monoclonal antibodies bind to the same, or a closelyrelated, epitope.

Still another way to determine whether a monoclonal antibody has thespecificity of a monoclonal antibody of the invention is to pre-incubatethe monoclonal antibody of the invention with the target molecule withwhich it is normally reactive, and then add the monoclonal antibodybeing tested to determine if the monoclonal antibody being tested isinhibited in its ability to bind the target molecule. If the monoclonalantibody being tested is inhibited then, in all likelihood, it has thesame, or functionally equivalent, epitopic specificity as the monoclonalantibody of the invention.

An additional way to determine whether a monoclonal antibody has thespecificity of a monoclonal antibody of the invention is to determinethe amino acid residue sequence of the complementarity determiningregions (CDRs) of the antibodies in question. Antibody molecules havingidentical, or functionally equivalent, amino acid residue sequences intheir CDRs have the same binding specificity. Methods for sequencingpolypeptides are well known in the art.

The immunospecificity of an antibody, its target molecule bindingcapacity, and the attendant affinity the antibody exhibits for theepitope, are defined by the epitope with which the antibodyimmunoreacts. The epitope specificity is defined at least in part by theamino acid residue sequence of the variable region of the heavy chain ofthe immunoglobulin that comprises the antibody, and in part by the lightchain variable region amino acid residue sequence. Use of the terms“having the binding specificity of” or “having the binding preferenceof” indicates that equivalent monoclonal antibodies exhibit the same orsimilar immunoreaction (binding) characteristics and compete for bindingto a preselected target molecule. Humanized monoclonal antibodies offerparticular advantages over murine monoclonal antibodies, particularlyinsofar as they can be used therapeutically in humans. Specifically,human antibodies are not cleared from the circulation as rapidly as“foreign” antigens, and do not activate the immune system in the samemanner as foreign antigens and foreign antibodies. Methods of preparing“humanized” antibodies are generally well known in the art, and canreadily be applied to the antibodies of the present invention. Thus, theinvention provides, in one embodiment, a monoclonal antibody of thepresent invention that is humanized by grafting to introduce componentsof the human immune system without substantially interfering with theability of the antibody to bind antigen. Humanized antibodies can alsobe produced using animals engineering to produce humanized antibodies,such as those available from Medarex of Annandale, N.J. (mice) andAbgenix, Inc., of Fremont, Calif. (mice). The use of a molecular cloningapproach to generate antibodies, particularly monoclonal antibodies, andmore particularly single chain monoclonal antibodies, is also provided.

The production of single chain antibodies has been described in the art,as for example in U.S. Pat. No. 5,260,203, the contents of which areherein incorporated by reference. For this, combinatorial immunoglobulinphagemid or phage-displayed libraries are prepared from RNA isolatedfrom the spleen of the immunized animal, and phagemids expressingappropriate antibodies are selected by panning on endothelial tissue.This approach can also be used to prepared humanized antibodies. Theadvantages of this approach over conventional hybridoma techniques arethat approximately 10⁴ times as many antibodies can be produced andscreened in a single round, and that new specificities are generated byH and L chain combination in a single chain, which further increases thechance of finding appropriate antibodies. Thus, an antibody suitable foruse in the present invention, or a “derivative” of an antibody of thepresent invention pertains to a single polypeptide chain bindingmolecule which has binding specificity and affinity substantiallysimilar to the binding specificity and affinity of the light and heavychain aggregate variable region of an antibody described herein.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species(scFv), one heavy- and one light-chain variable domain can be covalentlylinked by a flexible peptide linker such that the light and heavy chainscan associate in a “dimeric” structure analogous to that in a two-chainFv species. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The present invention also includes the use of a CCR5 antagonist fortreating or preventing stress response in a subject in need thereof. Thepresent invention further includes the use of a CCR5 antagonist in themanufacture of a medicament for treating or preventing stress responsein a subject in need thereof. Embodiments of these uses are the uses asjust described incorporating one or more of the embodiments, aspects andfeatures of any one or more of the previously described treatment andprevention methods of the invention. Thus, embodiments of theabove-described uses include uses in which the CCR5 antagonist is acompound of Formula (I), or is a compound of Formula (II), or isCompound A; and/or the subject is a warm-blooded vertebrate, or is aprimate, or is a human; and/or the subject is other than a grafttransplant patient, or is a cardiac surgery patient; and/or the stressresponse comprises hyperthermia (or hypothermia); and/or the use isinhibiting hyperthermia (or hypothermia).

The present invention also includes a method for treating or preventingstress response in a subject in need thereof which comprisesadministering to the subject a therapeutically effective amount of aCCR5 antagonist and a therapeutically effective amount of animmunosuppressive agent. The CCR5 antagonist can be administered before,concurrently with, or after administration of the immunosuprressiveagent. The term “immunosuppressive agent” refers to compounds which caninhibit an immune response. In one embodiment, the immunosuppressiveagent is selected from the group consisting of calcineurin inhibitors(e.g., cyclosporin A, FK506), IL2 signal transduction inhibitors (e.g.,rapamycin), glucocorticoids (e.g., prednisone, dexamethasone,methylprednisolone, prednisolone), nucleic acid synthesis inhibitors(e.g., azathioprine, mercaptopurine, mycophenolic acid), antibodies tolymphocytes or antigen-binding fragments thereof (e.g., OKT3, anti-EL2receptor, anti-CD52), and lymphocyte sequestrants (e.g., FTY720). Inanother embodiment, the immunosuppressive agent is a calcineurininhibitor. In an aspect of this embodiment, the calcineurin inhibitor iscyclosporin A. In still another embodiment, the immunosuppressive agentis a lymphocyte sequestrant. Additional embodiments of this method isthe method as first described or as described in one of the foregoingembodiments incorporating one or more of the embodiments, aspects andfeatures of any one or more of the previously described treatment andprevention methods of the invention. Thus, for example, one embodimentof this method is the method in which the subject is other than a grafttransplant patient.

The present invention also includes a method of treating or preventing adisorder characterized by the activity of at least one pro-inflammatorycytokine selected from the group consisting of IL1, IL6 and TNF (e.g.,at least one cytokine selected from IL1 and IL6), in a mammal (e.g., aprimate, especially a human) in need of such treatment or prevention,which comprises administering to the mammal a CCR5 modulator in anamount effective to inhibit endogenous production of the cytokine. Inone embodiment of this method, the CCR5 modulator comprises a CCR5antagonist. In an aspect of this embodiment, the CCR5 antagonistcomprises a small molecule organic compound, a polypeptide, or anantibody. Features of this aspect include the method wherein the CCR5modulator is a compound of Formula (I) or a pharmaceutically acceptablesalt or individual diastereomer thereof, a compound of Formula (II) or apharmaceutically acceptable salt thereof, or Compound A or apharmaceutically acceptable salt thereof, each as heretofore defined anddescribed. In another embodiment, the disorder being treated orprevented is selected from the group consisting of post-surgicalinflammatory response, sepsis, septic shock, and acute respiratorydistress syndrome (ARDS). Aspects and features of this embodimentinclude aspects and features described above for the previousembodiment.

The present invention also includes a method of treating a post-traumainflammatory response in a subject undergoing or having undergone amultiple trauma associated with a high risk of sepsis or ARDS, whichcomprises administering to the subject a therapeutically effectiveamount of a CCR5 antagonist. Exemplary multiple traumas pelvic ormultiple long bone fracture, massive blood loss, multiple unit bloodtransfusion, prolonged hypotension/shock, and pulmonary contusion. Inone embodiment of this method, the CCR5 antagonist comprises a smallmolecule organic compound, a polypeptide, or an antibody. Aspects ofthis embodiment include the method wherein the CCR5 antagonist is acompound of Formula (I) or a pharmaceutically acceptable salt orindividual diastereomer thereof, a compound of Formula (II) or apharmaceutically acceptable salt thereof, or Compound A or apharmaceutically acceptable salt thereof, each as heretofore defined anddescribed.

The term “post-surgical inflammatory response” as used herein refers toany disease, symptom, or pathological condition resulting from anexcessive or unregulated inflammatory response following surgery thatcan be attributed to the induction of at least one of thepro-inflammatory cytokines IL1, IL6 and TNF.

The term “sepsis” (also known in the art as systemic inflammatoryresponse syndrome (SIRS)) refers to a syndrome in which immunemediators, such as pro-inflammatory cytokines, produced or released inresponse to, for example, microbial invasion (e.g., by gram-negativebacteria with concomitant endotoxin infusion), injury (e.g., multiplelong bone fracture) or other insults (e.g., burns), induce an acutestate of inflammation which leads to abnormal homeostasis, organ damageand eventually to lethal shock. Individuals with sepsis typicallyexhibit fever, tachycardia, tachypnea, leukocytosis, and a localizedsite of infection. Microbiologic cultures from blood or the infectionsite are frequently, but not always, positive. “Septic shock” can occursubsequent to sepsis and refers to the condition in which pathogenicmicroorganisms, typically gram-negative bacteria, or their toxins arepresent in the blood or in other tissues during infection. Its symptomstypically include a drop in blood pressure, fever, diarrhea, widespreadblood clotting in various organs, and ultimately organ failure. “Acuterespiratory distress syndrome” (ARDS) refers to lung-failure relatedpathophysiology that is typically the first of the multiple organfailures that characterize the terminal phase of sepsis and septicshock.

The present invention also includes a method of inhibiting endogenousproduction of at least one pro-inflammatory cytokine selected from thegroup consisting of IL1, IL6, and TNF (e.g., at least one cytokineselected from IL1 and IL6), which comprises administering to a mammal inneed of such inhibition a CCR5 modulator in an amount effective toinhibit production of the cytokine. In one embodiment of this method,the CCR5 modulator comprises a CCR5 antagonist. In an aspect of thisembodiment, the CCR5 antagonist comprises a small molecule organiccompound, a polypeptide, or an antibody. Features of this aspect includethe method wherein the CCR5 modulator is a compound of Formula (I) or apharmaceutically acceptable salt or individual diastereomer thereof, acompound of Formula (II) or a pharmaceutically acceptable salt thereof,or Compound A or a pharmaceutically acceptable salt thereof, each asheretofore defined and described.

The present invention further includes a method for monitoring theeffectiveness of treatment of a subject (typically a mammal, preferablya primate, more preferably a human) suffering from an acute inflammatoryresponse, said treatment comprising administration of a CCR5 modulator,wherein the method comprises:

-   -   (A) obtaining a pre-administration sample (e.g., a serum or        tissue sample) from the subject prior to administration of the        CCR5 modulator and determining the level of expression or        activity of a pro-inflammatory cytokine selected from the group        consisting of IL1, IL6 and TNF (e.g., a cytokine selected from        IL1 and IL6) in the pre-administration sample;    -   (B) obtaining a post-administration sample from the subject        subsequent to administration of the CCR5 modulator and        determining the level of expression or activity of the        pro-inflammatory cytokine; and    -   (C) comparing the level of cytokine expression or activity of        the post-administration sample with the level of cytokine        expression or activity of the pre-administration sample.

An embodiment of this method is the method as just described, whichfurther comprises:

-   -   (D) adjusting the administration of the CCR5 modulator to        increase or decrease the level of cytokine expression or        activity; and    -   (E) repeating steps (A), (B), and (C).

In an aspect of this method and its foregoing embodiment, the CCR5modulator comprises a CCR5 antagonist. In another aspect, the CCR5modulator comprises a CCR5 antagonist which comprises a small moleculeorganic compound, a polypeptide, or an antibody. The CCR5 antagonist canbe a compound of Formula (I) or a pharmaceutically acceptable salt orindividual diastereomer thereof, a compound of Formula (II) or apharmaceutically acceptable salt thereof, or Compound A or apharmaceutically acceptable salt thereof, each as heretofore defined anddescribed.

The level of expression or activity of IL1, IL6, and TNF can bedetermined from serum, plasma, or whole blood samples in accordance withprocedures described in Casey et al., Ann. Intern. Med. 1993, 119(8):771-778, and in Bolke et al. Shock 2001, 16(5): 334-9. Cytokine levelscan also be determined by flow cytometry using, for example, BectonDickinson's Cytometric Bead Array Technology.

The present invention also includes a method for determining theefficacy of a CCR5 modulator in correcting an abnormal level of apro-inflammatory cytokine selected from the group consisting of IL1, IL6and TNF (e.g., a cytokine selected from IL1 and IL6) in a subject inneed of such correction, which comprises:

-   -   (A) administering an amount of the CCR5 modulator to the        subject; and    -   (B) determining the level of the cytokine in the subject        following administration of the CCR5 modulator, wherein a change        in the cytokine level toward a normal level is a measure of the        efficacy of the modulator.

An embodiment of this method is a method for determining the efficacy ofa CCR5 antagonist in reducing an abnormally high level of apro-inflammatory cytokine selected from the group consisting of IL1, IL6and TNF (e.g., a cytokine selected from IL1 and IL6) in a subject inneed of such reduction, which comprises:

-   -   (A) administering an amount of the CCR5 antagonist to the        subject; and    -   (B) determining the level of the cytokine in the subject        following administration of the CCR5 antagonist, wherein a        reduction in the cytokine level toward a normal level is a        measure of the efficacy of the antagonist.

In an aspect of this embodiment, the CCR5 antagonist comprises a smallmolecule organic compound, a polypeptide, or an antibody. In stillanother aspect, the CCR5 antagonist can be a compound of Formula (I) ora pharmaceutically acceptable salt or individual diastereomer thereof, acompound of Formula (II) or a pharmaceutically acceptable salt thereof,or Compound A or a pharmaceutically acceptable salt thereof, each asheretofore defined and described.

The term “abnormal level” (which may also be referred to as an “aberrantlevel”) refers to a level of a pro-inflammatory cytokine (IL1, IL6 orTNF) which is measurably different (whether higher or lower) from thelevel of the cytokine in the healthy subject. As already noted above,because the normal level of cytokine can vary from one subject to thenext, it is preferred to determine the normal cytokine level for theparticular subject prior to exposure to an insult (e.g., prior to aplanned insult such as surgery). Alternatively, the normal cytokinelevel can be equated to the average value obtained or known for a groupof similarly situated healthy individuals.

The activity of agents (e.g., small molecule organics, polypeptides,antibodies) as chemokine receptor CCR5 modulators can be determinedusing methods known in the art. More particularly, the activity of anagent as a CCR5 receptor modulator can be determined using a suitablescreen (e.g., high through-put assay). For example, an agent can betested in an extracellular acidification assay, calcium flux assay,ligand binding assay or chemotaxis assay. Suitable assays are described,for example, in Hale et al., Bioorg. & Med. Chem. Letters 2001, 11:1437-1440; Hesselgesser et al., J. Biol. Chem. 1998, 273 (25):15687-15692; WO 98/18826 and WO 98/02151, the disclosures of which areincorporated herein by reference. Also suitable for assessing theactivity of agents as CCR5 modulators are the assays described in WO01/78707, the disclosure of which is incorporated herein by reference.

The active agent(s) (i.e., a chemokine receptor CCR5 modulator andoptionally one or more additional therapeutic agents) can beadministered orally, parenterally (including intravenous, intramuscular,or intrasternal injection, or infusion techniques), by subcutaneousadministration (e.g., injection), by inhalation spray, by buccaldelivery, by surgical implantation, or rectally, in the form of a unitdosage of a pharmaceutical composition containing a therapeuticallyeffective amount of the modulator (e.g., a small organic molecule) andconventional non-toxic pharmaceutically-acceptable carriers, adjuvantsand vehicles. The particular mode of drug administration used inaccordance with the methods of the present invention depends on variousfactors, including but not limited to the severity of the condition tobe treated and mechanisms for metabolism or removal of the drugfollowing administration. Oral and parenteral administration aregenerally preferred, however.

Suitable formulations for injection include aqueous and non-aqueoussterile solutions that can contain antioxidants, buffers, bacteriostats,bactericidal antibiotics and solutes that render the formulationisotonic with the bodily fluids of the intended recipient; and aqueousand non-aqueous sterile suspensions, which can include suspending agentsand thickening agents.

For oral administration, the compositions can take the form of, forexample, tablets or capsules prepared by conventional techniques withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets can be coated by methods known in the art. For example, a CCR5antagonist can be formulated in combination with hydrochlorothiazide,and as a pH stabilized core having an enteric or delayed release coatingwhich protects the CCR5 antagonist until it reaches the colon.

Liquid preparations for oral administration can take the form of, forexample, solutions, syrups or suspensions, or they can be presented as adry product for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional techniqueswith pharmaceutically acceptable additives such as suspending agents(e.g., sorbitol syrup, cellulose derivatives or hydrogenated ediblefats); emulsifying agents (e.g. lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations can alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration can be suitablyformulated to give controlled release of the active compound. For buccaladministration the compositions can take the form of tablets or lozengesformulated in a conventional manner.

For rectal administration, the agents (e.g., CCR5 antagonist andoptionally one or more additional therapeutic agents) can be formulatedas suppositories or retention enemas containing conventional suppositorybases such as cocoa butter or other glycerides).

The agents can also be formulated as creams or lotions, or transdermalpatches.

The amount of the agent (e.g., CCR5 receptor modulator) administered tothe subject can be varied so as to administer an amount that iseffective to achieve the desired therapeutic response. The selecteddosage level and frequency of administration thereof will depend upon avariety of factors including the activity of the composition, choice offormulation, the route of administration, combination with other drugsor treatments, the severity of the condition being treated, and thephysical condition (e.g., health, age, sex, weight, diet) and priormedical history of the subject being treated. In one embodiment, aminimal dose is administered, and dose escalated in the absence ofdose-limiting toxicity to a minimally effective amount. In anotherembodiment, the agent is administered in an amount of from about 0.001to about 1000 mg/kg of body weight of the subject per day in a singledose or in divided doses. A preferred dosage range is from about 0.01 toabout 500 mg/kg body weight per day in a single dose or divided doses.In still another embodiment, the agent is administered in a range offrom about 0.1 to 100 mg per day to an adult human.

It is understood that when two or more agents are administered, theagents can be administered concurrently or sequentially in either order,with a suitable mode of administration and dosage form selected foreach. When the agents are administered at different times, the timeinterval between administrations is suitably selected such that there isan overlap in the efficacy and activity of each of the agents, wherebyan additive or synergistic benefit is provided. The person of ordinaryskill in the art can determine the appropriate order of and timeinterval between administration of the agents and can also determine theappropriate dosage level and form for each agent.

For purposes of prevention, the CCR5 modulator is suitably administeredbefore the insult (when such insult is planned such as elective surgery)or during or after its occurrence but before the stress responsemanifests itself. In the case of surgical insult, the time foradministration is chosen such that the agent will provide efficacy andpharmacological activity at the time the insult is scheduled to occur.The agent is suitably administered within about about 24 hours (e.g.,from about 1 to about 12 hours) prior to insult, and is typicallyadministered within about 8 hours (e.g., from about 0.5 to about 4hours) prior to surgery. The timing will depend upon thepharmacokinetics of the agent, mode of administration, the physicalcondition of the patient, and other factors related to administrationfrequency and dosage level described earlier.

Additional guidance regarding formulation and dose can be found in U.S.Pat. No. 5,326,902; U.S. Pat. No. 5,234,933; WO 93/25521; Berkow et al.(1997) The Merck Manual of Medical Information, Home ed. Merck ResearchLaboratories, Whitehouse Station, N.J.; Goodman et al. (1996) Goodman &Gilman's the Pharmacological Basis of Therapeutics, 9th ed. McGraw-HillHealth Professions Division, New York; Ebadi (1998) CRC Desk Referenceof Clinical Pharmacology. CRC Press, Boca Raton, Fla.; Katzung (2001)Basic & Clinical Pharmacology, 8th ed. Lange Medical Books/McGraw-HillMedical Pub. Division, New York; Remington et al. (1975) Remington'sPharmaceutical Sciences, 15th ed. Mack Pub. Co., Easton, Pa.; andSpeight et al. (1997) Avery's Drug Treatment: A Guide to the Properties,Choice, Therapeutic Use and Economic Value of Drugs in DiseaseManagement, 4th ed. Adis International, Auckland/Philadelphia; Duch etal. (1998) Toxicol Lett 100-101:255-263.

The following examples serve only to illustrate the invention and itspractice. The examples are not to be construed as limitations on thescope or spirit of the invention.

EXAMPLE 1 Fever Suppression after Cardiac Allotransplantation

This example shows fever suppression in a group of monkeys treated witha CCR5 antagonist after undergoing cardiac allotransplantation.Cynomolgus macaques (Macaca fascicularis), weighing 2.4-4.7 kg, andranging in estimated age from 2.2-5.5 years were selected as organrecipients. Donors were matched to recipients by AB blood typecompatibility as tested by New York University, Nelson Institute ofEnvironmental Medicine, Tuxedo, N.Y. MHC class II mismatch was assuredby a stimulation index (SI)>3 in paired animals in unidirectional mixedlymphocyte reaction (MLR) using potential donor lymphocytes asstimulators and potential recipient peripheral blood mononuclear cells(PBMCs) as responders. Animals were matched by assigning pairs withmaximal MLR response within groups of blood type compatible animals.

Anesthesia: Anesthesia was induced with intramuscular ketamine (10mg/kg) (Fort Dodge Animal Health, Fort Dodge, Iowa) and glycopyrrolate(0.01 mg/kg IM) (Fort Dodge Animal Health, Fort Dodge, Iowa) andmaintained with propofol (i.e., 2,6-diisopropylphenol) (AbbottLaboratories, Chicago, Ill.) to effect a surgical plane of anethesia. Inanimals with established venous access, anesthesia was induced withpropofol.

Donor operation: After systemic heparinization (70 units/kg, Elkins-SinnInc., Cherry Hill, N.J.) and collection of donor blood duringequivolemic saline infusion, diastolic arrest of the donor heart wasinduced with University of Wisconsin organ preservation solution (15-20cc/kg, 4° C.) via the aortic root. The heart was explanted and prepared:The mitral valve was rendered incompetent surgically, to prevent leftventricular distention, and an atrial septal defect was created byexcising the fossa ovalis, to allow blood entering the left heartthrough the thebesian circulation to transit to and be ejected from theright heart. The left atrium was oversewn, and the supra vena cava (SVC)and inferior vena cava (IVC) ligated.

Recipient operation: All recipient animals underwent traditionalnon-working intraabdominal cardiac allograft transplantation using asingle clamp technique, wherein the donor aorta was anastomosedend-to-side to the infrarenal abdominal aorta, and the donor pulmonaryartery was anastomosed to the adjacent vena cava. Heparin (70 Units/kg)(a Unit is equivalent to 3 mg/kg of body weight) was administered to therecipient prior to clamp placement. A silicon central venous catheterwas introduced via the internal jugular vein, and tunneled to exitbetween the scapulae. Catheters were attached to swivel connectionsystems, and animals placed in protective cloth jackets incorporating aswiveling system which protected venous access.

Postoperative analgesia consisted of intramuscular buprenorphine 0.01mg/kg (Reckitt & Colman Pharm., Richmond, Va.) and ketoprofen 2 mg/kg(Fort Dodge Animal Health, Fort Dodge, Iowa).

Graft function and body temperature were assessed twice daily bymonitoring with a fully implantable telemetry device (Data SciencesInternational, St. Paul, Minn.) (electrocardiogram (EKG), rightventricular pressure and animal body temperature) implanted at the timeof transplantation. Confirmatory abdominal ultrasounds were performed atthe time of protocol biopsies and whenever an examiner appreciateddecreased contractility, if EKG voltage or rate (<120 beats per minute)was decreased, or if developed pulse pressure (delta P) in the graft wasdecreased. Cardiac biopsies were performed by protocol on postoperativedays 4, 7, 14, through a laparotomy incision using a core biopsy needle.Graft failure was defined as loss of palpable graft activity, withultrasound confirmation of weak or absent myocardial contractility.Failed grafts were explanted promptly and examined histologically.

Blood draws were performed from a peripheral site on the days ofsurgical intervention to monitor complete blood counts and bloodchemistry for eventual drug related toxicity or side effects.

Three animals received Compound A as monotherapy at a dose of 5 mg/kg,two animals received Compound A as monotherapy at a dose of 10 mg/kg,and three animals received an equivalent volume of saline (control).Compound A was administered intravenously twice daily at 12 hourintervals (i.e., b.i.d.) starting at transplant. Two other animalsreceived Compound A at 5 mg/kg b.i.d. combined with a subtherapeuticamount cyclosporin A (CsA), and a further two animals (control) receivedCsA only. The CsA was dosed by daily intramuscular injection. The doseon the first day was 12.5 mg/kg, then 10 mg/kg daily for 7 days, then 5mg/kg daily for 7 days, then 2.5 mg/kg/day until graft rejection oranimal sacrifice. In one instance, an animal initially treated withCompound A alone (10 mg/kg b.i.d.) was rescued on day 9 with bolussteroids (40 mg/kg on day 1; 20 mg/kg SID on days 2 and 3) and thentreated with combined therapy for 43 days. On day 52, CsA treatment wasstopped, and the animal remained on a twice daily regimen of Compound A(10 mg/kg). Vigorous graft function persisted to day 83, 30 days afterstopping CsA.

Post transplantation, the animals were individually housed in stainlesssteel metal caging, maintained at 22° C. with 12-hour light/dark cycles.Tap water was available ad libidum, and the monkeys were fed commercialprimate chow and fruit.

The febrile response characteristic of the first three days afterallograft implantation in control animals and animals treated withconventional immunosuppression (i.e., CsA) was not seen in CCR5antagonist-treated animals. In the three control animals that receivedsaline infusions, the average temperature was consistently above 38° C.Similarly in the two animals treated with CsA alone, fever was observedin one of the animals. In total, fever was observed in 4 of the 5animals not receiving Compound A, and was typically recurrent in thoseanimals. In contrast, the average temperature in six of the sevenanimals treated with Compound A was below 37.5° C., and individualtemperature elevations reached 38.5° C. on any occasion in only 3 of theanimals. In summary, six of seven monkeys treated with a CCR5 antagonistdid not develop a fever (i.e., a temperature greater than 38.5° C.)while recovering from the transplantation procedure, and had an averagetemperature about one degree lower than the control monkeys receiving noimmunosuppressive therapy and one half degree lower than animals inwhich acute rejection was prevented by CsA therapy. In addition, incontrast to the five control animals, the CCR5 antagonist-treatedmonkeys were observed not to exhibit malaise and to behave as if theyhad not had surgery. The treated monkeys also did not exhibit anysymptoms of abdominal tenderness that would otherwise have beenexpected, despite major biochemical perturbations including increasedcreatinine and bilirubin (as determined by analysis of the serumchemistries) that would normally have been associated with discomfortand malaise. These findings indicate that CCR5 plays a previouslyunrecognized role in the acute phase inflammatory response.

Double-label immunofluorescence microscopy using antibodies specific forCD3, CD68, CD11b, CCR5 and CXCR3 was used to characterize leukocytepopulations infiltrating acutely rejecting (days 4, 6) cynomolguscardiac allografts. CCR5 and CXCR3 are prominent onmonocytes/macrophages and to a lesser extent on T-cells in rejectingcynomolgus heart allografts. Co-localization of CCR5 and CXCR3 to thesame cells was common, particularly on the D68 monocyte/macrophagepopulation. Infiltration of CCR5+ cells was inhibited in associationwith CCR5 blockade. Graft failure was identified by a decrease in heartrate and ST elevation (associated with ischemia) on EKG and a decreasein arterial line pressure. Graft survival was prolonged from 6±0.4 days(n=7) to 8.3±0.6 days (n=3) (p=0.05) (n=3 refers to the animals whoreceived 5 mg/kg Compound A monotherapy) despite pharmacokinetics datafor Compound A (i.e. trough concentrations determined by LC-MS/MSfollowing solid phase extraction of plasma using Waters OASIS 96-wellextraction plate (30 mg)) that suggested full receptor coverage may nothave been achieved at trough drug concentrations; i.e., the levels ofCompound A fell below the target range at trough of 100 nM, aconcentration which corresponded to about 90% receptor occupancy. (Note:n=7 refers to the data for the 3 control (i.e., saline) monkeys in thisexperiment plus data for 4 animals from a historic database that werescored as controls in which cyno cardiac allografts were rejected in theabsense of immunosupression in a manner identical to the three controlsin this experiment.)

Administration of Compound A in combination with CsA had a much greatereffect than the use of CsA alone as this regimen extended survival from13.5±1.5 days (n=2) for CsA monotherapy to greater than 21 days and 44days.

Remarkably, the animal initially treated with Compound A alone, rescuedon day 9 with steroids, and then treated with combined therapy for 43days had vigorous graft function at day 78, 25 days after stopping CsA.

EXAMPLE 2 Cytokine Determination

IL1β and IL6 can be measured using the DuoSet ELISA development kitsfrom R&D Systems (Minneapolis, Minn.), following the directions of themanufacturer. Briefly, plates are coated with capture antibody [mouseanti-human IL1β (2 μg/ml) or mouse anti-human IL6 (4 μg/ml)] anddetection antibodies are used at 100 ng/ml (biotinylated goat anti-humanIL1β) or 200 ng/ml (biotinylated goat anti-human IL6). Standards areprepared with recombinant protein (3.9-250 pg/ml for IL1β and 4.7-300pg/ml for IL6). Streptavidin conjugated to horseradish peroxidase andH₂O₂/tetramethylbenzidine are used for color development. Opticaldensity (OD) is measured at 450 nm, with correction at 570 nm.

TNFα is measured by ELISA using the mouse-anti-human TNFα antibody BC7(Cell Sciences, Inc., Norwood, Mass.) as the capture antibody at 2.5μg/ml, and biotinylated goat anti-human TNFα BAF210 (R&D Systems) as thedetection antibody at 800 ng/ml. A standard is prepared with recombinantprotein (10-10,000 pg/ml). Streptavidin conjugated to horseradishperoxidase and H₂O₂/tetramethylbenzidine is used for color development.OD is measured at 450 nm, with correction at 570 nm.

Serum samples were taken from the cynomolgus monkeys in Example 1 atvarious time points pre- and post-transplantation. The samples werediluted 1:2, in PBS/0.1% BSA/0.05% Tween 20. (PBS=phosphate bufferedsaline; BSA=bovine serum albumin.) IL6 levels (pg/ml) in pre and postheterotopic heart transplant in serum from the cynomolgus monkeystreated with Compound A were determined obtained in accordance with theabove-described procedure. The results are in Table 1 as follows: TABLE1 Day 0 Day 0 Day Animal Pre Post 4 Day 7 Day 8 Day 14 Day 21 M308 4.663.7 35.1 48.4 24.3 M347 2.8 421.4 12.7 15 6 7.5 M376 2.5 374.9 11.4 116.3 10.3 M627 3.4 106.2 8.8 19.6M308 received Compound A monotherapy at 10 mg/kg/day b.i.d..M347 and M376 received 5 mg/kg Compound A plus CsA tapered.M627 received Compound A monotherapy at 10 mg/kg/day for 9.5 days, thenwas rescued before acute organ rejection by bolus steroids, followed bya tapered CsA regimen.

The results show that a dramatic increase in IL6 levels occurredimmediately following the transplantation, followed by a substantialreduction thereafter. These results indicate that the administration ofa CCR5 antagonist such as Compound A can substantially reduce orsuppress the level of pro-inflammatory cytokine associated with an acuteinflammatory response.

EXAMPLE 3 Plasma Cytokine Levels in Transplanted Cynomologous Monkeys

Serum and plasma samples taken from the cynomologous monkeys in Example1 on the day of explant (i.e., the day of graft rejection) were analyzedusing the BD Pharmingen Cytometric Bead Array Human Inflammation Kit,which can measure IL1-β, IL6, IL8, IL10, IL12p70 and TNF-α. Reagents todetect IL6, IL8 and TNF-α have been shown by the manufacturer to crossreact with rhesus and cynomologus monkey proteins. At the time of theassay, reagents to measure IL1-beta and IL12p70 had not yet been testedfor cross reactivity to non-human primate proteins.

Samples were assayed in the manner described in the manufacturer'sprotocol booklet; i.e., “Human Inflammation Kit—Instruction Manual” (BDBiosciences, Cat. No. 551811, pdf copy available atwww.bdbiosciences.com). Briefly, assay standards were reconstituted withassay diluent and diluted in two fold steps by serial dilution. Capturebeads were mixed and distributed into an appropriate number of 12×75 mmpolystyrene tubes. Standards or samples were added to the tubes andincubated in the dark at room temperature for 1.5 hours. The incubatedtubes were washed once and phycoerythrin (PE)-labeled detection antibodyreagent was added. Samples were again incubated in the dark at roomtemperature for 1.5 hours. After one final wash, samples wereresuspended in 300 uL of wash buffer. Flow cytometer data was acquiredusing a Becton Dickinson FACSCalibur and analyzed with Becton DickinsonCBA Software.

The results for the IL6 assay are shown in Table 2. TABLE 2 Explant IL6Animal Treatment¹ Day (pg/mL) M308 Compound A monotherapy at 8 58 10mg/kg/day b.i.d. M627 Compound A monotherapy at 85 20 10 mg/kg/dayb.i.d. M347 Compound A (5 mg/kg) plus tapered CsA 44 no sample M376Compound A (5 mg/kg) plus tapered CsA 21 22 M324 tapered CsA 15 170 M634tapered CsA 13 206¹Refer to Example 1 for a more detailed description of the treatmentprovided to the monkeys following surgery. Note in particular that M627was initially treated with Compound A alone (10 mg/kg b.i.d.), wasrescued on day 9 with bolus steroids, treated with combined therapy# for 43 days, and then from day 52 until explant received Compound Amonotherapy at 10 mg/kg b.i.d.

The data in Table 2 show that IL6 cytokine levels are suppressed inmonkeys treated with Compound A at the time of graft rejection relativeto monkeys treated only with CsA. It is likely that the IL6 levels wouldhave been higher in the monkeys in the absence of any treatment. Thedata indicate that the administration of a CCR5 antagonist such asCompound A can substantially reduce or suppress IL6 levels associatedwith an inflammatory response. Analogous data obtained for IL8 cytokinelevels did not indicate suppression of IL8 levels for Compound A-treatedmonkeys relative to CsA-only treated monkeys at the time of graftrejection.

IL-12p70, TNF-alpha and IL-10 were detected at very low levels in allsamples. IL-1β was detected only in some samples. There were nosignificant changes in the levels of these cytokines betweentransplanted and control monkeys or within treatment groups.

EXAMPLE 4 Effect of Compound A on Cytokine Expression in HumanMacrophages

Human monocytes were isolated white blood cells concentrated from unitsof blood (i.e., leukopaks) from normal donors. To enhance CCR5expression, the monocytes were cultured under suspension conditions inTeflon jars for 2 days in media supplemented with 12% fetal bovineserum. 10⁶ cells were seeded and allowed to attach for over 2 hours in6-well tissue culture plates. Cells were pre-incubated with eitherdimethylsulfoxide (DMSO; vehicle control) or Compound A at variousconcentrations for 1 hour, after which RANTES (the CCR5 agonist/ligand;Pepro Tech Inc.) was added at 250 nM, or, alternatively, media was addedas a control. The cells were incubated at 37° C. for 24 hours, afterwhich the media were removed and centrifuged to precipitate thenon-adherent cells. Both the adhered and non-adhered cells were lysedand subjected to mRNA isolation, and the mRNA isolate was analyzed forcytokine expression by quantitative real time PCR (TaqMan).

The relative mRNA-fold induction of cytokine gene expression in theRANTES-stimulated macrophages was calculated relative to day 2non-stimulated, non-treated controls (i.e. fold induction=1 on day 2 forcontrols), as well as fold mRNA induction relative to vehicle (DMSO) (0nM) control RANTES stimulated cells. A summary of the results of theTaqMan analysis is as follows:

-   -   (i) RANTES stimulation increased mRNA expression of IL1-beta and        IL6, whereas other cytokines such as TNF-α and IFN-γ were        unaffected.    -   (ii) Compound A was able to diminish IL1-β and IL6 up-regulation        significantly.

(iii) The inhibitory effect by Compound A on IL1-β and IL6 mRNAexpression in the macrophages was dose-dependent, as shown by theresults in Table 3. TABLE 3 Compound A IL1-beta (% (nM) inhibition) IL6(% inhibition) 0 0 0 3 37.6 (±32.1) 43 10 50.1 (±25.8) 16.1 (±6.3) 3059.0 (±26.8) 65.9 (±6.3) 100 76.8 (±21.8) 67.1 300 80.6 (±12.6)  85.9(±12.9)The values are mean ± SD (if applicable). No induction of TNF-α or IFN-γwas observed so as to give percent inhibition levels.

References have been made throughout this application to variouspublished documents in order to more fully describe the state of the artto which this invention pertains. All of these documents not previouslyincorporated by reference and are hereby incorporated by reference intheir entireties.

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, thepractice of the invention encompasses all of the usual variations,adaptations and/or modifications that come within the scope of thefollowing claims.

1. A method of treating or preventing stress response in a subject in need thereof, which comprises administering a therapeutically effective amount of a CCR5 antagonist to the subject. 2-3. (canceled)
 4. The method according to claim 1, wherein the subject is other than a graft transplant patient.
 5. The method according to claim 1, wherein the stress response is stress response to surgery.
 6. The method according to claim 1, wherein the subject is a cardiac surgery patient.
 7. The method according to claim 1, wherein the therapeutically effective amount of CCR5 antagonist administered to the subject is an amount effective to inhibit endogenous production of one or more pro-inflammatory cytokines selected from the group consisting of IL1 and IL6. 8-10. (canceled)
 11. A method of treating or preventing hyperthermia in a subject in need thereof, which comprises administering a therapeutically effective amount of a CCR5 antagonist to the subject. 12-13. (canceled)
 14. The method according to claim 11, wherein the subject is other than a graft transplant patient.
 15. The method according to claim 11, wherein the stress response is surgical hyperthermia.
 16. The method according to claim 11, wherein the subject is a cardiac surgery patient.
 17. The method according to claim 11, wherein the therapeutically effective amount of CCR5 antagonist administered to the subject is an amount effective to inhibit endogenous production of one or more pro-inflammatory cytokines selected from the group consisting of IL1 and IL6. 18-41. (canceled)
 42. A method of inhibiting endogenous production of at least one pro-inflammatory cytokine selected from the group consisting of IL1 and IL6, which comprises administering to a mammal in need of such inhibition a CCR5 modulator in an amount effective to inhibit production of the cytokine.
 43. The method according to claim 42, wherein the CCR5 modulator comprises a CCR5 antagonist.
 44. A method for monitoring the effectiveness of treatment of a subject suffering from an acute inflammatory response, said treatment comprising administration of a CCR5 modulator, wherein the method comprises: (A) obtaining a pre-administration sample from the subject prior to administration of the CCR5 modulator and determining the level of expression or activity of a pro-inflammatory cytokine selected from the group consisting of IL1 and IL6 in the pre-administration sample; (B) obtaining a post-administration sample from the subject subsequent to administration of the CCR5 modulator and determining the level of expression or activity of the pro-inflammatory cytokine; and (C) comparing the level of cytokine expression or activity of the post-administration sample with the level of cytokine expression or activity of the pre-administration sample.
 45. The method according to claim 44, which further comprises: (D) adjusting the administration of the CCR5 modulator to increase or decrease the level of cytokine expression or activity; and (E) repeating steps (A), (B), and (C).
 46. The method according to claim 45, wherein the CCR5 modulator comprises a CCR5 antagonist. 47.-52. (canceled) 